Fiber-coupled, high-speed, angled-dual-axis optical coherence scanning microscopes

ABSTRACT

This invention provides an angled-dual-axis optical coherence scanning microscope comprising a fiber-coupled, high-speed angled-dual-axis confocal scanning head and a vertical scanning unit. The angled-dual-axis confocal scanning head is configured such that an illumination beam and an observation beam intersect optimally at an angle θ within an object and the scanning is achieved by pivoting the illumination and observation beams jointly using a high-speed scanning element, thereby producing an arc-line scan. The vertical scanning unit causes the angled-dual-axis confocal scanning head to move towards or away from the object. Optical coherence detection is employed to provide temporal gating, thus detecting mostly single-scattered light and preventing multiple-scattered light from dominating the signal when imaging in a scattering medium. By incorporating MEMS scanning mirrors and fiber-optic components, the angled-dual-axis optical coherence scanning microscope can be miniaturized to provide a particularly powerful tool for in vivo medical imaging applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to the following U.S. patent applications,all of which are herein incorporated by reference: “Fiber-coupled,High-speed, Integrated, Angled-Dual-Axis Confocal Scanning MicroscopesEmploying Vertical-Section Scanning” of Michael J. Mandella, Mark H.Garrett, and Gordon S. Kino, Ser. No. 09/628,118; and “Fiber-coupled,Angled-Dual-Axis Confocal Scanning Microscopes for Imaging in aScattering Medium” of Michael J. Mandella, Mark H. Garrett, and GordonS. Kino, Ser. No. 09/627,363.

FIELD OF THE INVENTION

This invention relates generally to confocal scanning microscopy andoptical coherence microscopy. More specifically, it relates tofiber-based optical coherence microscopy systems incorporating a novel,fiber-coupled, angled-dual-axis confocal scanning microscope.

BACKGROUND ART

The advent of fiber optics and laser technology has brought a renewedlife to many areas of conventional optics. Confocal microscopes, forexample, have enjoyed higher resolution, more integrated structure, andenhanced imaging capability. Consequently, confocal microscopes havebecome increasingly powerful tools in a variety of applications,including biological and medical imaging, optical data storage andsemiconductor applications. The original idea of confocal microscopytraces back to the work of Marvin Minsky. Described in his seminal U.S.Pat. No. 3,013,467 is a system in which an illumination beam passesthrough a pinhole, traverses a beamsplitter, and is focused by anobjective to a focal volume within an object. An observation beam thatemanates from the local volume is in turn converged by the sameobjective lens, reflected by its second encounter with the beamsplitter,and passes through a second pinhole to a photo detector. The geometry ofthis confocal arrangement is such that only the light beam originatingfrom the focal volume is able to pass through the second pinhole andreach the photo detector, thus effectively discriminating all otherout-of-focus signals.

Contemporary confocal microscopes tend to adopt one of two basicconfocal geometries. In the transmission arrangement using twoobjectives, one objective focuses an illumination beam from a pointsource onto a focal volume within an object and another objectivecollects an observation beam that emanates from a confocal overlappingvolume (within the focal volume). Whereas in the so-called “reciprocal”reflection arrangement, a single objective plays a dual role of focusinglight on the object and collecting the light emanated from the object.In either case, the confocal arrangement enables the confocal microscopeto attain a higher resolution and sharper definition than a conventionalmicroscope, because out-of-focus signals are rejected. This uniqueability has made confocal microscopes particularly useful tools in theexamination of biological specimens, since they can view a specificlayer within a sample and avoid seeing other layers, the so-called“optical sectioning”. Confocal microscopy techniques are also exploitedto provide a spatial filter in many applications.

The transmission confocal microscope typically employs two separatelenses: one serves as the illumination objective and the other as theobservation objective. The single objective in the “reciprocal”arrangement can also be a single lens, in either simple or compoundform. In order to image a thin layer about a few micrometers thickwithin a sample, however, the numerical aperture (NA) of the objectivelenses must be large, so as to provide adequate resolution particularlyin the axial direction. This generally results in a short workingdistance, which is undesirable in practice.

A great deal of ingenuity has accordingly been devoted to improving theaxial resolution of confocal microscopes without using high NA lenses. Aparticularly interesting approach is to spatially arrange two separateillumination and observation objective -lenses, or the illumination andobservation beam paths, in such a way that the illumination beam and theobservation beam intersect at an angle theta (θ) at the focal points, sothat the overall point-spread function for the microscope, i.e., theoverlapping volume of the illumination and observation point-spreadfunctions results in a substantial reduction in the axial direction. Aconfocal microscope with such an angled, dual-axis design is termed aconfocal theta microscope, or an angled-dual-axis confocal microscope,hereinafter. The underlying principle as well as the advantages ofconfocal theta microscopy are described in US Patent Application,“Fiber-coupled, High-speed, Integrated, Angled-Dual-Axis ConfocalScanning Microscopes Employing. Vertical Cross-Section Scanning” ofMichael J. Mandella, Mark H. Garrett, and Gordon S. Kino, Ser. No.09/628,118, filed on Jul. 8, 2000, incorporated herein by reference forall purposes.

More specifically, Ser. No. 09/628,118 discloses an angled-dual-axisconfocal scanning microscope comprising an angled-dual-axis confocalscanning head mechanically coupled to a vertical scanning unit. Theangled-dual-axis confocal scanning head is configured such that theillumination and observation beams intersect optimally at an angle θwithin an object and the scanning is achieved by pivoting theillumination and observation beams jointly using a single scanningelement, thereby producing an arc-line scan. The vertical scanning unitfurther causes the angled-dual-axis confocal scanning head to movetowards or away from the object, whereby a succession of arc-line scansthat progressively deepen into the object is produced, providing atwo-dimensional vertical cross-section scan of the object. The verticalscanning unit also comprises a compensation means, for keeping theoptical path lengths of the illumination and observation beams unchangedso to ensure the optimal intersection of the illumination andobservation beams in the course of vertical scanning. This novelscanning mechanism, along with the integrated structure of theangled-dual-axis confocal scanning head and the coupling of opticalfibers, enables this angled-dual-axis confocal scanning microscope toperform fast and high resolution scanning over a large transverse fieldof view, while maintaining a workable working distance. The integrationof optical fibers and silicon fabrication technology further rendersthis angled-dual-axis confocal scanning microscope integrity,flexibility, scalability, and maneuverability, as desired in manyapplications.

For example, one of the applications the aforementioned angled-dual-axisconfocal scanning microscope is particularly suited for is opticalcoherence microscopy (OCM), which effectively filters outmultiple-scattered photon noise, thus providing high sensitivity andlarge dynamic range of detection when imaging in a scattering medium.Although great stride has been made in improving the sensitivity andimaging capabilities of optical coherence microscopy, as exemplified byU.S. Pat. No. 6,201,608 B1, commonly assigned to the same inventors asthe current application, optical coherence microscopy has yet to reachits full potential of high resolution and fast scanning, as required inbiological and medical applications, particularly in vivo imaging oflive tissue which is constantly in motion. Two of the prior art methodsof obtaining high axial resolution in an OCM apparatus involve the useof either a large NA objective lens, or the use of a femto-second pulsedlaser with a very short coherence length. These methods are described byWang et al. in “High Speed, full field optical coherence microscopy”,Proceedings of The SPIE Conference on Coherence Domain Optical Methodsin Biomedical Science and Clinical Applications III, San Jose, Calif.,January 1999, pp. 204-212, and by Drexler et al. in “In vivoultrahigh-resolution optical coherence tomography”, Optics Letters,21(17), pp. 1221-1223, 1999, all incorporated herein by reference. Theprimary disadvantage of using a high NA is the limited field of view inwhich diffraction-limited performance is obtained during high speedtransverse scanning. High cost and intricacy of femto-second lasers makethe second approach undesirable for a practical instrument.

Hence, there is a need in the art for a new way of applying thetechniques of optical coherence microscopy that overcomes thelimitations of the prior art methods.

OBJECTS AND ADVANTAGES

Accordingly it is a principal object of the present invention to providean angled-dual-axis optical coherence scanning microscope that achieves

a) high axial resolution;

b) large transverse field of view;

c) long working distance;

d) high-speed vertical cross-section scanning;

e) higher sensitivity and larger dynamic range;

f) improved contrast when imaging in a scattering medium;

g) flexibility and scalability, and

h) simple and low cost construction.

These and other objects and advantages will become apparent from thefollowing description and accompanying drawings.

SUMMARY OF THE INVENTION

This invention provides an angled-dual-axis confocal scanningmicroscope. A first embodiment of the angled-dual-axis confocal scanningmicroscope of the present invention comprises an angled-dual-axisconfocal scanning head and a vertical scanning means. Theangled-dual-axis confocal scanning head further comprises a first end ofa first single-mode optical fiber serving as a point light source, anangled-dual-axis focusing means, an arc-line scanning means, and a firstend of a second single-mode optical fiber serving as a point lightdetector.

From the first end of the first optical fiber an illumination beamemerges. The angled-dual-axis focusing means serves to focus theillumination beam to a diffraction-limited illumination focal volumealong an illumination axis within an object. The angled-dual-axisfocusing means further receives an observation beam emanated from anobservation focal volume along an observation axis within the object,and focuses the observation beam to the first end of the second opticalfiber. The angled-dual-axis focusing means is designed such that theillumination axis and the observation axis intersect at an angle θwithin the object, thereby the illumination and observation focalvolumes intersect optimally at a confocal overlapping volume. Thearc-line scanning means, preferably in the form of a single scanningelement such as a silicon micro-machined scanning mirror, is positionedsuch that it receives the illumination beam from the angled-dual-axisfocusing means and directs the illumination beam to the object. Thearc-line scanning means also collects the observation beam emanated fromthe object and passes the observation beam to the angled-dual-axisfocusing means. The arc-line scanning means is further capable ofpivoting the illumination and observation beams in such a way that theillumination and observation axes remain intersecting at a fixed angle θand that the confocal overlapping volume moves along an arc-lineperpendicular to both the illumination and observation axes within theobject, thereby producing an arc-line scan.

The vertical scanning means, in the form of a vertical scanning unit,comprises a vertical translation means and a compensation means. Thevertical translation means is mechanically coupled to theangled-dual-axis confocal scanning head, such that it causes theangled-dual-axis confocal scanning head to move towards or away from theobject, whereby a succession of arc-line scans that progressively deepeninto the object is produced, providing a two-dimensional verticalcross-section scan of the object. The compensation means keeps theoptical path lengths of the illumination and observation beamssubstantially unchanged, so as to ensure the optimal intersection of theillumination and observation focal volumes in the course of verticalscanning. Such a compensation mechanism is also crucial for performingoptical coherence microscopy.

Altogether, the first embodiment of the angled-dual-axis confocalscanning microscope of the present invention is designed such that it iscapable of performing vertical cross-section scanning in a line-by-linefashion with enhanced axial (i.e., vertical) resolution and greaterspeed, while maintaining a workable working distance and a large fieldof view. In applications where a three-dimensional volume image of theobject is desired, the object may be further moved incrementally along atransverse direction as each vertical cross-section scan is completed. Aplurality of vertical cross-section images thus generated can beassembled into a three-dimensional volume image of a region within theobject.

In a second embodiment of the angled-dual-axis confocal scanningmicroscope of the present invention, an angled-dual-axis confocal headis mechanically coupled to a vertical scanning means and a transversescanning means. The angled-dual-axis confocal head comprises a first endof a first single-mode optical fiber serving as a point light source, anangled-dual-axis focusing means, and a first end of a second single-modeoptical fiber serving as a point light detector.

From the first end of the first optical fiber an illumination beamemerges. The angled-dual-axis focusing means serves to focus theillumination beam to a diffraction-limited illumination focal volumealong an illumination axis within an object. The angled-dual-axisfocusing means further receives an observation beam emanated from anobservation focal volume along an observation axis within the object,and focuses the observation beam to the first end of the second opticalfiber. The angled-dual-axis focusing means is designed such that theillumination axis and the observation axis intersect at an angle θ withthe object, thereby the illumination and observation focal volumesintersect optimally at a confocal overlapping volume. The verticalscanning means, in the form of a vertical scanning unit, comprises avertical translation means and a compensation means. The verticaltranslation means is mechanically coupled to the angled-dual-axisconfocal head, such that it causes the angled-dual-axis confocal head tomove towards or away from the object, whereby producing a vertical scanthat deepens into the interior of the object. The compensation meanskeeps the optical path lengths of the illumination and observation beamssubstantially unchanged, thereby ensuring the optimal intersection ofthe illumination and observation focal volumes in the course of verticalscanning. Moreover, the transverse scanning means, in the form of atransverse stage, serves to translate the object relative to theangled-dual-axis confocal head along transverse directions perpendicularto the vertical direction, thereby providing a transverse scan.

As such, the second embodiment of the angled-dual-axis confocal scanningmicroscope of the present invention is capable of performing verticalscans and transverse scans in various ways. By assembling an assortmentof the vertical and/or transverse scans in a suitable manner,two-dimensional transverse and/or vertical cross-section images of theobject can be obtained. A three-dimensional volume image of the objectcan also be accordingly constructed.

It is to be understood that the term “emanating” as used in thisspecification is to be construed in a broad sense as covering any lighttransmitted back from the object, including reflected light andscattered light. It should be also understood that when describing theintersection of the illumination and observation beams in thisspecification, the term “optimal” means that the illumination andobservation focal volumes (i.e., the main lobes of the illuminationbeam's point-spread function and the observation beam's point-spreadfunction) intersect in such a way that their respective centerssubstantially coincide and the resulting overlapping volume hascomparable transverse and axial extents. This optimal overlapping volumeis termed “confocal overlapping volume” in this specification.

In an angled-dual-axis confocal scanning microscope of the presentinvention, the angled-dual-axis focusing means generally comprises anassembly of beam focusing, collimating, and deflecting elements. Suchelements can be selected from the group of refractive lenses,diffractive lenses, GRIN lenses, focusing gratings, micro-lenses,holographic optical elements, binary lenses, curved mirrors, flatmirrors, prisms and the like. A crucial feature of the angled-dual-axisfocusing means is that it provides an illumination axis and anobservation axis that intersect at an angle θ. The optical fibers can besingle-mode fibers, multi-mode fibers, birefrigent fibers, polarizationmaintaining fibers and the like. Single-mode fibers are preferable inthe present invention, for the ends of single-mode fibers provide anearly point-like light source and detector.

The aforementioned arc-line scanning means typically comprises anelement selected from the group consisting of scanning mirrors,reflectors, acousto-optic deflectors, electro-optic deflectors,mechanical scanning mechanisms, and Micro-Electro-Mechanical-Systems(MEMS) scanning micro-mirrors. A preferred choice for the arc-linescanning means is a flat pivoting mirror, particularly a siliconmicro-machined scanning mirror for its compact and light-weightconstruction. Moreover, the arc-line scanning means is placed betweenthe angled-dual-axis focusing means and the object to be examined. Thisenables the best on-axis illumination and observation point-spreadfunctions to be utilized throughout the entire angular range of anarc-line scan, thereby providing greater resolution over a largertransverse field of view, while maintaining diffraction-limitedperformance. Such an arrangement is made possible by taking advantage ofthe longer working distance rendered by using relatively lower NAfocusing elements or lenses in the angled-dual-axis focusing means.

A distinct advantage of the angled-dual-axis confocal scanningmicroscope of the present invention is that the scanning is achieved bypivoting both the illumination and observation beams, as opposed tomoving either the object or the microscope's objective lenses in theprior art confocal theta scanning microscopes, which can be quitecumbersome to implement and adversely limits the precision of scanning.

Another important advantage of the angled-dual-axis arrangement of thepresent invention is that since the observation beam is positioned at anangle relative to the illumination beam, scattered light along theillumination beam does not easily get passed into the observation beam,except where the beams overlap. This substantially reduces scatteredphoton noise in the observation beam, thus enhancing the sensitivity anddynamic range of detection. This is in contrast to the direct couplingof scattered photon noise between the illumination and observation beamsin a transmission or reciprocal confocal microscope, due to thecollinear arrangement between the beams. Moreover, by using low NAfocusing elements (or lenses) in an angled-dual-axis confocal scanningsystem of the present invention, the illumination and observation beamsdo not become overlapping until very close to the focus. Such anarrangement prevents additional scattered light in the illumination beamfrom directly “jumping” to the observation beam, hence further filteringout multiple-scattered photon noise in the observation beam.Unfortunately, this arrangement does not eliminate multiple-scatteredphoton noise that originates within the observation beam. The presentinvention employs a temporal gating technique, to filter out this sourceof noise. Altogether, the angled-dual-axis confocal scanning system ofthe present invention has much lower noise and is capable of providingmuch higher contrast when imaging in a highly scattering medium than theprior art confocal systems.

A further advantage of the present invention is that the entireangled-dual-axis confocal scanning head in the first embodiment, or theangled-dual-axis confocal head in the second embodiment, can be mountedon a silicon substrate etched with precision V-grooves which hostvarious optical elements. Such an integrated device offers a high degreeof integrity, maneuverability, scalability, and versatility, whilemaintaining a workable working distance and a large field of view. Inparticular, a micro-optic version of an integrated, angled-dual-axisconfocal scanning head (or angled-dual-axis confocal head) of thepresent invention can be very useful in biological and medical imagingapplications, e.g., endoscopes and hand-held optical biopsy instruments.

All in all, the angled-dual-axis contocal scanning microscope of thepresent invention provides high resolution scanning with greaterprecision and faster speed, while maintaining a workable workingdistance and a large field of view. The angled-dual-axis confocalscanning microscope of the present invention further advantageouslyexploits the flexibility, scalability and integrity afforded by opticalfibers and silicon micro-machining techniques, rendering it a highlyversatile and modular device.

The present invention provides an angled-dual-axis optical coherencescanning microscope incorporating the aforementioned angled-dual-axisconfocal scanning microscope. An exemplary embodiment of theangled-dual-axis optical coherence scanning microscope of the presentinvention comprises an angled-dual-axis confocal scanning microscope asdescribed above (e.g., in its first or second exemplary embodiment), alight source, a beam-splitting means, a reference optical fiber, and abeam-combining means. The beam-splitting means is in opticalcommunication with the light source and the angled-dual-axis confocalscanning microscope, such that it diverts a portion of an output beamemitted from the light source to the first optical fiber of theangled-dual-axis confocal scanning microscope and a remainder of theoutput beam to the reference optical fiber, thereby creating anillumination beam and a reference beam from the same parent beam. Anobservation beam collected by the angled-dual-axis confocal scanningmicroscope is delivered by way of the second optical fiber of theangled-dual-axis confocal scanning microscope, and is further combinedwith the reference beam at the beam-combining means to generate coherentinterference.

In the embodiment described above, the beam-combining means can be inthe form of a fiber-optic coupler, at which the reference and secondoptical fibers are joined and the reference and observation beams arecombined. Balanced detection scheme can be accordingly utilized. Thesystem may further comprise a frequency shifting means optically coupledto the first or second optical fiber of the angled-dual-axis confocalscanning microscope, such that the frequency of the observation beam isshifted relative to the frequency of the reference beam. Alternatively,the frequency shifting means can be optically coupled to the referenceoptical fiber, such that the frequency of the reference beam is shiftedrelative to the frequency of the observation beam. In either case,coherent interference between the reference and observation beams ismodulated at a heterodyne beat frequency given by the relative frequencyshift between the reference and observation beams, allowing for moresensitive heterodyne detection. Moreover, an adjustable optical delaydevice may be coupled to the reference optical fiber, the first orsecond optical fiber, so as to maintain coherent interference betweenthe reference and observation beams at the fiber-optic coupler wherethey are combined. In applications where the light source has a shortcoherence length, the optical delay device can be adjusted such thatmostly single-scattered light in the observation beam is coherent withthe reference beam at the fiber-optic coupler and multiple-scatteredlight in the observation beam, which traverses over a longer opticalpath length, does not contribute to the coherent interference, thereforeproviding further filtering of multiple-scattered light upon detection.To enhance the signal-to-noise ratio of detection, an optical amplifiercan be coupled to the second optical fiber, so as to boost up the powerof the observation beam returning from the object. An amplifiedobservation beam has an additional advantage of rendering fasterscanning rates and consequently higher pixel rates without appreciableloss in the signal-to-noise ratio, because a shorter integration timeper pixel of an image is required in data collection. The implementationof balanced detection in this case allows subtraction of the amplifiernoise, since preponderance of the spontaneous emission of the opticalamplifier would not occur at the heterodyne beat frequency describedabove.

The light source in the above embodiment can be an optical fiberamplifier, semiconductor optical amplifier, a fiber laser, asemiconductor laser, a diode-pumped solid state laser, or a broadbandOCT light source. The light source may be polarized, or unpolarized. Thebeam-splitting means can be a fiber-optic coupler, such as an evanescentwave coupler or a fused fiber coupler. Various optical fibers, such asthe first, second, and reference optical fibers, are preferablysingle-mode fibers, for single-mode fibers have the advantage ofsimplicity and automatic assurance of the mutual spatial coherence ofthe observation and reference beams upon mixing and detection.

In one case where polarized light is provided by the light source andthe beam-splitting means is a polarizing beamsplitter, the orientationof the beamsplitter relative to the polarization of light emitted fromthe light source can be used to control the ratio of optical powerbetween the illumination and reference beams. Furthermore, the first,second, and reference optical fibers are preferably polarizationmaintaining (PM) fibers, to control the polarizations of theillumination, observation and reference beams throughout the entiresystem. The optical coupling between the polarized light source and thepolarizing beamsplitter is also preferably by way of a third PM fiber.In this case, the reference and observation beams can be brought intothe same polarization by rotating either the reference or second opticalfiber before joining them at the fiber-optic coupler. Alternatively, apolarization rotation means, such as a Faraday rotator, can be coupledto either the reference optical fiber or the second optical fiber, suchthat the reference and observation beams have substantially the samepolarization when combined.

The aforementioned embodiment can also be used to provide specificinformation pertaining to the polarization state of light emanated froma polarization-altering, such as a birefrigent-scattering, medium. Manybiological tissues, such as tendons, muscle, nerve, bone, cartilage andteeth, exhibit birefrigence due to their linear or fibrous structure.Birefrigence causes the polarization state of light to be altered (e.g.,rotated) in a prescribed manner upon refection. Thus, by detectinginduced changes in the polarization state of light reflected from abirefrigent-scattering medium, image representing birefrigent (or otherpolarization-altering) “scatterers” can be obtained. In such a case, thepolarizing beamsplitter produces an illumination beam withP-polarization and a reference beam with orthogonal S-polarization froma polarized beam emitted from the light source. An observation beamreflected from a birefirgent-scattering (or polarization-altering)sample carries both P-polarization and S-polarization, where thepresence of S-polarization is resulted from the birefrigent (or otherpolarization-altering) “scatterers” in the sample. When the referenceand observation beams are combined at the fiber-optic coupler, only theobservation beam with S-polarization interferes coherently with thereference beam that has only S-polarization. Consequently, the amplitudeof resulting heterodyne beat frequency signal corresponds only to theamplitude of reflectance of light with S-polarization, hence providingan image representing birefrigent (or other polarization-altering)“scatterers” in the sample.

In applications where the observation beam with P-polarization is ofgreater interest, a polarization rotation means, such as a rotatablefiber connector or a Faraday rotator, can be coupled to the reference PMfiber in the above embodiment, such that the polarization of thereference beam is rotated by 90-degree, resulting a reference beam withP-polarization. Upon combining the reference and observation beams inthis case, only the observation beam with P-polarization interferescoherently with the reference beam that now has only P-polarization.Consequently, the amplitude of resulting heterodyne beat frequencysignal measures only the amplitude of reflectance of light withP-polarization.

Moreover, in applications where both P-polarization and S-polarizationof the observation beam are of interest, a first auxiliary polarizingbeamsplitter can be optically coupled to the second PM fiber of theangled-dual-axis confocal scanning microscope, serving to separateP-polarization and S-polarization of the observation beam by routingthem to fourth and fifth PM fibers, respectively. A polarizationrotation means, such as a rotatable fiber connector or a Faradayrotator, can be optically coupled to the reference PM fiber and servesto rotate the polarization of the reference beam by 45-degree, thusrendering the reference beam with both P-polarization andS-polarization. The polarization-rotated reference beam is thendelivered to a second auxiliary polarizing beamsplitter, which in turnseparates P-polarization and S-polarization of the reference beam byrouting them to sixth and seventh PM fibers, respectively. The systemmay further comprise a frequency shifting means optically coupled to thefirst or second PM fiber, such that the frequency of the observationbeam is shifted relative to the reference beam. The fourth and sixth PMfibers can be further joined by a first auxiliary fiber-optic coupler,where the observation and reference beams with S-polarization arecoherently combined and the amplitude of resulting heterodyne beatfrequency signal corresponds to the amplitude of reflectance of lightwith S-polarization. Similarly, the fifth and seventh PM fibers can bejoined by a second auxiliary fiber-optic coupler, at which theobservation and reference beams with P-polarization are coherentlycombined and the amplitude of resulting heterodyne beat frequency signalmeasures the amplitude of reflectance of light with P-polarization. Assuch, the exemplary system thus described can be utilized to provide animage pertaining to the birefrigent-scattering (or otherpolarization-altering) regions in a sample with enhanced contrast.

It should be noted that in the above exemplary cases involving polarizedlight, a polarization maintaining fiber-optic coupler can bealternatively used with the polarized light source. A polarizationrotation means may be optically coupled to the reference PM fiber forrotating the polarization of the reference beam, so as to select thedesired polarization of the observation beam. Moreover, an unpolarizedlight source along with a polarizing beamsplitter can be used to providea polarized illumination beam and a polarized reference beam withorthogonal polarization. A disadvantage of using an unpolarized lightsource is, however, that the ratio of optical power between theillumination and reference beams cannot be efficiently adjusted to bestsuit a particular application.

The present invention further provides an alternative embodiment of anangled-dual-axis optical coherence microscope, comprising a light sourceequipped with dual output-ports, an angled-dual-axis confocal scanningmicroscope as previously described, and a reference optical fiber. Afirst output-port of the light source is optically coupled to the firstoptical fiber of the angled-dual-axis confocal scanning microscope,transmitting an illumination beam. A second output-port of the lightsource is optically coupled to the reference optical fiber, providing areference beam. An observation beam collected by the angled-dual-axisconfocal scanning microscope is delivered by way of the second opticalfiber, and is in turn combined with the reference beam such thatcoherent interference is produced for detection.

In the aforementioned embodiment, the reference and second opticalfibers may be joined by a fiber-optic coupler, to provide for a balanceddetection scheme. The system further comprises a frequency shiftingmeans optically coupled to the first or second optical fiber of theangled-dual-axis confocal scanning microscope, such that the frequencyof the observation beam is shifted relative to the frequency of thereference beam. Alternatively, the frequency shifting means can beoptically coupled to the reference optical fiber, such that thefrequency of the reference beam is shifted relative to the frequency ofthe observation beam. In either case, coherent interference between thereference and observation beams is modulated at a beat frequency, suchthat heterodyne balanced detection can be utilized. Moreover, anadjustable optical delay device may be coupled to either the referenceoptical fiber, or the second optical fiber, to maintain coherentinterference between the reference and observation beams at thefiber-optic coupler where they are combined. To enhance thesignal-to-noise ratio of detection, an optical amplifier can be coupledto the second optical fiber, so as to boost up the power of theobservation beam.

The light source in the above embodiment is preferably a short coherencelength source, such as the type commonly used for optical coherencetomography applications. The light source can also be an optical fiberamplifier, a semiconductor optical amplifier, a fiber laser, asemiconductor laser, or a diode-pumped solid state laser, equipped withdual output-ports. Various optical fibers, such as the first, second,and reference optical fibers, are preferably single-mode fibers, forsingle-mode fibers have the advantage of simplicity and automaticassurance of the mutual spatial coherence of the observation andreference beams upon mixing and detection. If polarized light isprovided by the light source, the first, second, and reference opticalfibers are preferably polarization maintaining fibers. To ensure thatthe reference and observation beams have the same polarization upondetection, either the reference or second optical fiber can be rotatedby an appropriate amount before they are joined at the fiber-opticcoupler.

The angled-dual-axis optical coherence microscope of the presentinvention, as the above exemplary embodiments demonstrate, offers theadvantages of enhanced axial resolution while maintaining a workableworking distance, fast and high-precision scanning over a large field ofview, while attaining higher sensitivity and larger dynamic range ofdetection provided by the optical coherence technique. It alsoadvantageously exploits the flexibility, scalability, ruggedness, andlow cost afforded by optical fibers. As such, the angled-dual-axisoptical coherence microscope of the present invention is particularlysuited for applications in which high resolution, high contrast imagingand fast scanning are required, such as in vivo imaging of live tissuefor performing optical biopsies in many medical applications.

The novel features of this invention, as well as the invention itself,will be best understood from the following drawings and detaileddescription.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D shows several exemplary embodiments of an angled-dual-axisconfocal scanning head according to the present invention;

FIGS. 2A-2C depict three exemplary embodiments of an angled-dual-axisconfocal scanning microscope according to the present invention;

FIGS. 3A-3C illustrate simplified schematic diagrams of first, secondand third embodiments of an angled-dual-axis optical coherence scanningmicroscope; and

FIG. 4 depicts a simplified schematic diagram of a fourth embodiment ofan angled-dual-axis optical coherence scanning microscope of the presentinvention.

DETAILED DESCRIPTION

Although the following detailed description contains many specificdetails for the purposes of illustration, anyone of ordinary skill inthe art will appreciate that many variations and alterations to thefollowing details are within the scope of the invention. Accordingly,the exemplary embodiment of the invention described below is set forthwithout any loss of generality to, and without imposing limitationsupon, the claimed invention.

FIGS. 1A-1D depict several exemplary embodiments of an angled-dual-axisconfocal scanning head according to the present invention. Depicted inFIG. 1A is a first exemplary embodiment of an angled-dual-axis confocalscanning head of the present invention. Confocal scanning head 100, byway of example, comprises a first end 101 of a first optical fiber 103serving as a point light source; a first end 102 of a second opticalfiber 104 serving as a point light detector; an angled-dual-axisfocusing means in the form of a lens assembly consisting of firstcollimating lens 105, second collimating lens 106, illumination lens107, observation lens 108, and two beam-aligning elements in the form offirst 45-degree mirror 109 and second 45-degree mirror 110; an arc-linescanning means in the form of a single scanning mirror 111, which can bepivoted about axis 122, and a silicon substrate 112. First collimatinglens 105, illumination lens 107, and first mirror 109 are mounted onfirst V-groove 113 etched on substrate 112. Similarly, secondcollimating lens 106, observation lens 108, and second mirror 110 aremounted on second V-groove 114 etched on substrate 112. First opticalfiber 103 and second optical fiber 104 are affixed to cylindricalferrules 115 and 116 respectively, which are in turn mounted onV-grooves 113 and 114 respectively, such that fiber ends 101 and 102 areheld in their respective positions.

In operation, an illumination beam 117 emerges from first end 101 offirst optical fiber 103 and is directed to first collimating lens 105.The collimated beam is then passed onto and focused by illumination lens107. The focused beam is further deflected by first mirror 109 toscanning mirror 111, which in turn directs the beam to adiffraction-limited illumination focal volume (see FIG. 1B) within anobject 120.

An observation beam 118 emanated from a diffraction-limited, confocaloverlapping volume 121 is first collected by scanning mirror 111, thendirected to second mirror 110, which in turn deflects the beam toobservation lens 108. Observation beam 118 is further collimated byobservation lens 108 and then focused by second collimating lens 106 tofirst end 102 of second optical fiber 104. Scanning mirror 111 ispositioned such that illumination beam 117 and observation beam 118intersect optimally at an angle (θ) within object 120.

FIG. 1B provides a more detailed illustration of how illumination beam117 and observation beam 118 are arranged to intersect within object 120in FIG. 1A. Illumination beam 117, directed by scanning mirror 111, isfocused to an illumination focal volume 99 oriented along anillumination axis 97 within object 120. Observation beam 118 emanatesfrom an observation focal volume 98 oriented along an observation axis96 within object 120 is received by scanning mirror 111. Illuminationaxis 97 and observation axis 96 are directed to intersect at angle θ,such that illumination focal volume 99 and observation focal volume 98intersect optimally at confocal overlapping volume 121. Athree-dimensional x-y-z coordinate system is provided to describe thespatial extents of confocal overlapping volume 121, where the origin ofthe coordinate system is set at the center of confocal overlappingvolume 121. The z-axis defines the axial (or vertical) direction,whereas x-axis and y-axis (pointing out of the page) represent twoorthogonal transverse directions.

It is to be understood that the term “emanating” as used in thisspecification is to be construed in a broad sense as covering any lighttransmitted back from the object, including reflected light andscattered light.

In the present invention, various optical elements areaberration-corrected, and single-mode optical fibers are used to providea point light source and detector. Accordingly, illumination focalvolume 99 and observation focal volume 98 described above arediffraction-limited, defined by the main lobes of the illuminationbeam's point-spread function and observation beam's point-spreadfunction. Confocal overlapping volume 121 is likewisediffraction-limited, determined by an optimal overlapping of the mainlobes of the illumination beam's point-spread function and theobservation beam's point-spread function, as illustrated in FIG. 1B.

Ser. No. 09/628,118, incorporated herein by reference, discloses amathematical model for calculating the point-spread functions of twooptimally intersecting focused beams, such as illumination beam 117 andobservation beam 118 exemplified in FIG. 1B, thereby providing anestimate of the three-dimensional extents of the resulting confocaloverlapping volume, e.g., confocal overlapping volume 121. As theexemplary model calculations and accompanying graphs in Ser. No.09/628,118 indicate, the confocal overlapping volume displaysGaussian-like characteristics in x, y, and z directions, diminishingrapidly and monotonically (i.e., there are no additional side-bands)with increasing distance from the center of the two-beam overlappingregion. Such characteristics define a sharp resolution in both the axialas well as transverse directions. As a way of example, for anangled-dual-axis confocal system with an intersecting angle θ (=2α)equal to 60° and NA of the objective lenses about 0.25, thecorresponding axial resolution is approximately 2.8 microns, and thetransverse resolutions about 1.6 and 1.4 microns, respectively.

Now referring back to FIG. 1A, by rotating about axis 122 at a variableangle φ, scanning mirror 111 is further capable of pivoting illuminationbeam 117 and observation beam 118 jointly in such a way thatillumination beam 117 and observation beam 118 remain intersectingoptimally at angle θ and confocal overlapping volume 121 at theintersection of the two beams moves along an arc-line within object 120,thereby producing an arc-line scan.

It should be noted that an important characteristic of the arc-line scandescribed above is that the relative spatial orientation betweenillumination beam 117 and observation beam 118 stays fixed in the courseof the entire scan, once the two beams are arranged to intersect in anoptimal manner initially. This is in distinct contrast to the prior artconfocal theta scanning systems, where the illumination and observationbeams need to be separately adjusted at each scan point, in order toensure an optimal intersection. Consequently, the scans performed byangled-dual-axis confocal scanning head 100 of the present invention areinherently of higher precision and faster speed, and are also lesslaborious to carry out. Another notable feature of angled-dual-axisconfocal scanning head 100 of the present invention is that theillumination and observation beam paths can be exchanged, withoutaffecting its performance.

FIG. 1C shows a second exemplary embodiment of an angled-dual-axisconfocal scanning head of the present invention. In angled-dual-axisconfocal scanning head 150, an illumination reflective focusing element151 is implemented to replace first collimating lens 105, illuminationlens 107, and first mirror 109 in FIG. 1A, providing a dual function offocusing and deflecting illumination beam 117. Likewise, an observationreflective focusing element 152 is used to replace second mirror 110,observation lens 108, and second collimating lens 106 in FIG. 1A,providing a dual function of focusing and deflecting observation beam118. The remainder of angled-dual-axis confocal scanning head 150 sharesthe same components with angled-dual-axis confocal scanning head 100 inFIG. 1A. By way of example, illumination and observation reflectivefocusing elements 151, 152 are in the form of two diffractive lenseswith reflective coatings. The proper design of such reflectivediffraction lenses can be determined by commercially available computermodeling programs and is well known in the art of diffractive lensdesign. Illumination and observation reflective focusing elements 151,152 can alternatively be in the form of curved mirrors. In some cases ofusing curved mirrors such as ellipsoidal mirrors (each having two foci),it is desirable to place fiber ends 101, 102 at the respective firstfocal points of the two ellipsoidal mirrors serving as illumination andobservation focusing elements 151, 152, thereby focusing illuminationbeam 117 and observation beam 118 at the respective second focal pointsof focusing elements 151, 152. All in all, the overall operation ofangled-dual-axis confocal scanning head 150 is similar to the working ofangled-dual-axis confocal scanning head 100, as described above.

FIG. 1D depicts a simplified schematic illustration of a third exemplaryembodiment of an angled-dual-axis confocal scanning head of the presentinvention. Angled-dual-axis confocal scanning head 170 comprises a firstend 171 of a first optical fiber 173 serving as a point light source; afirst end 172 of a second optical fiber 174 serving as a point lightdetector; an angled-dual-axis focusing means in the form of a lensassembly consisting of first collimating lens 175, second collimatinglens 176, and illumination-observation lens 177; and an arc-linescanning means in the form of a single scanning mirror 178 having apivoting axis 185.

In operation, an illumination beam 179 emerges from first end 171 offirst optical fiber 173 and is directed to first collimating lens 175.The collimated beam is then passed onto and focused byillumination-observation lens 177. The focused beam reflects offscanning mirror 178 at first impingement spot 183, and is furtherdirected to a diffraction-limited illumination focal volume (not shownin FIG. 1D) within an object 182. An observation beam 180 emanated froma diffraction-limited, confocal overlapping volume 181 is first receivedby scanning mirror 178 at second impingement spot 184, further passedonto and collimated by illumination-observation lens 177. Observationbeam 180 is then focused by second collimating lens 176 to first end 172of second optical fiber 174. Scanning mirror 178 is positioned such thatillumination beam 179 and observation beam 180 intersect optimally at anangle (θ) within confocal overlapping volume 181, as illustrated in FIG.1B. By rotating about axis 185, scanning mirror 178 is further capableof pivoting illumination beam 179 and observation beam 180 jointly insuch a way that illumination beam 179 and observation beam 180 remainintersecting optimally at angle θ and confocal overlapping volume 181 atthe intersection of the two beams moves along an arc-line within object120, thereby producing an arc-line scan, as in the embodiment of FIG. 1Aor FIG. 1C.

It should be noted that in an angled-dual-axis arrangement of thepresent invention, as the above exemplary embodiments depict, since theobservation beam is positioned at an angle relative to the illuminationbeam, scattered light along the illumination beam does not easily getpassed into the observation beam, except where the beams overlap. Thissubstantially reduces scattered photon noise in the observation beam,thus enhancing the sensitivity and dynamic range of detection. This isin contrast to the direct coupling of scattered photon noise between theillumination and observation beams in a transmission or reciprocalconfocal microscope, due to the coilinear arrangement between the beams.Moreover, by using low NA focusing elements (or lenses) in anangled-dual-axis confocal scanning system of the present invention, theillumination and observation beams do not become overlapping until veryclose to the focus. Such an arrangement prevents additional scatteredlight in the illumination beam from directly “jumping” to theobservation beam, hence further filtering out multiple-scattered photonnoise in the observation beam. Unfortunately, this arrangement does noteliminate multiple-scattered photon noise that originates within theobservation beam. The present invention employs a temporal gatingtechnique, to filter out this source of noise. Altogether, theangled-dual-axis confocal scanning system of the present invention hasmuch lower noise and is capable of providing much higher contrast whenimaging in a highly scattering medium than the prior art confocalsystems.

FIGS. 1A, 1C-1D serve to illustrate only three of many embodiments of anangled-dual-axis confocal scanning head of the present invention. Ingeneral, the angled-dual-axis focusing means in an angled-dual-axisconfocal scanning head of the present invention comprises an assembly ofone or more elements for beam focusing, collimating, aligning, anddeflecting, as exemplified in FIGS. 1A, 1C-1D. Such elements can be inthe form of refractive lenses, diffractive lenses, GRIN lenses, focusinggratings, micro-lenses, holographic optical elements, binary lenses,curved mirrors, flat mirrors, prisms and the like. A crucial feature ofthe angled-dual-axis focusing means is that it provides an illuminationaxis and an observation axis that intersect at an angle, as illustratedin FIG. 1B. The arc-line scanning means in an angled-dual-axis confocalscanning head of the present invention generally comprises an elementselected from the group consisting of scanning mirrors, reflectors,acousto-optic deflectors, electro-optic deflectors, mechanical scanningmechanisms, and Micro-Electro-Mechanical-Systems (MEMS) scanningmicro-mirrors. A key feature is that the arc-line scanning means is inthe form of a single element, as opposed to two or more separatelyfunctioning scanning elements in prior art confocal scanning systems. Apreferred choice for the arc-line scanning means is a flat pivotingmirror, particularly a silicon micro-machined scanning mirror for itscompact and light-weight construction. (Note: to achieve fasterscanning, the scanning means can be in the form of two smaller coplanarpivoting mirrors, such as two silicon micro-machined scanning mirrors.Owing to their unique fabrication process, these mirrors can be operatedin substantially synchronous motion and constructed to co-rotate about acommon axis so as to scan illumination and observation beams in a wayfunctionally equivalent to a larger single scanning mirror.) Thefabrication processes of silicon scanning mirrors are described in U.S.Pat. Nos. 6,007,208, 6,057,952, 5,872,880, 6,044,705, 5,648,618,5,969,465, and 5,629,790. The optical fibers in an angled-dual-axisconfocal scanning head of the present invention can be single-modefibers, multi-mode fibers, birefrigent fibers, polarization maintainingfibers and the like. Single-mode fibers are preferable, however, for theends of single-mode fibers provide a nearly point-like light source anddetector.

A unique feature of the angled-dual-axis confocal scanning head of thepresent invention is that the arc-line scanning means is placed betweenthe angled-dual-axis focusing means and the object to be examined. Thisenables the best on-axis illumination and observation point-spreadfunctions to be utilized throughout the entire angular range of anarc-line scan, thereby providing greater resolution over a largertransverse field of view, while maintaining diffraction-limitedperformance. Such an arrangement is made possible by taking advantage ofthe longer working distance rendered by using relatively lower NAfocusing elements or lenses in the angled-dual-axis focusing means. Forexample, in the present invention, molded a spherical lenses with NAranging from 0.1 to 0.4 that are low cost and readily available in theart may be used. Such lenses have excellent on-axis aberrationcorrection, and are therefore diffraction-limited for on-axis focusingconditions. These lenses normally do not have diffraction-limitedperformance when focusing off-axis, and thus cannot be used in confocalscanning systems where off-axis performance is important. Such is thecase in prior art confocal scanning systems described in U.S. Pat. Nos.5,973,828 and 6,064,518, where the field of view is limited by theoff-axis performance of the objective lenses.

Moreover, the specific arrangements among various optical elements andoptical fibers in an angled-dual-axis confocal scanning head can bealtered in many ways without deviating from the principle and the scopeof the present invention. For instance, the use of collimating lensesand beam-aligning mirrors, such as those in FIG. 1A to help facilitatethe shaping and alignment of the illumination and observation beams, canbe optional and vary with the nature of practical applications. Otherauxiliary optical elements may also be implemented in anangled-dual-axis confocal scanning head of the present invention, toenhance the overall performance. All in all, a skilled artisan will knowhow to design an angled-dual-axis confocal scanning head in accordancewith the principle of the present invention, to best suit a givenapplication.

By integrating its constituent optical elements on a silicon substrate,as exemplified in FIGS. 1A, 1C by way of silicon fabrication techniques,the angled-dual-axis confocal scanning head of the present inventionrenders a high degree of integrity, maneuverability, scalability, andversatility.

Such a design also provides greater flexibility and higher precision inthe alignment of various optical elements. Although the particular wayof making an angled-dual-axis confocal scanning head of the presentinvention an integrated device is dictated by the nature of a givenapplication, a silicon substrate is generally preferred, for it is wellknown in the art that various V-grooves can be etched on silicon in avery precise manner, as demonstrated in U.S. Pat. Nos. 6,007,208 and5,872,880. The precision of the V-grooves provides an accurate andsecure optical alignment among various optical elements hosted by theseV-grooves, enabling the angled-dual-axis confocal scanning head thusconstructed to be a reliable and modular device. Using the embodiment ofFIG. 1A as a way of example, mirrors 109, 110 can be rotated about theirrespective axes and translated along V-grooves 113, 114 respectively, tofacilitate the optimal intersection of illumination and observationbeams 117, 118. Illumination and observation lenses 107, 108 can also betranslated along V-grooves 113, 114 respectively, to further facilitatethe optimal overlapping of illumination and observation focal volumes99, 98 as illustrated in FIG. 1B. Such alignment procedures can beperformed before affixing (e.g., by means of gluing) various opticalelements to their respective V-grooves. The scalability and relativelylow cost of silicon fabrication technology add further advantages tothis approach. For example, a micro-optic version of such an integratedangled-dual-axis confocal scanning head can be incorporated in miniaturesurgical devices, endoscopes, or other in situ devices, suitable formedical applications.

To provide a two-dimensional scan, an angled-dual-axis confocal scanninghead of the present invention can be mechanically coupled to a verticalscanning means in the form of a vertical scanning unit comprising avertical translation means and a compensation means. The verticaltranslation means causes the angled-dual-axis confocal scanning head tomove toward or away from the object and hence the illumination andobservation beams to probe further into the interior of the object,thereby producing a vertical scan. A two-dimensional verticalcross-section scan of the object is then obtained by assembling aplurality of arc-line scans that progressively deepen into the object.The compensation means keeps the optical path lengths of theillumination and observation beams substantially unchanged, therebyensuring the optimal intersection of the illumination and observationfocal volumes in the course of vertical scanning. This compensationfunction is also crucial for performing optical coherence microscopy.The combination of the angled-dual-axis confocal scanning head and thevertical scanning unit thus described constitutes a first embodiment ofan angled-dual-axis confocal scanning microscope, as depicted in FIG.2A. Angled-dual-axis confocal scanning microscope 200, by way ofexample, comprises an angled-dual-axis confocal scanning head (ADACSH)201 and a vertical translation means in the form of a movable carriage202. For the purpose of illustration, angled-dual-axis confocal scanninghead 201 is in a simplified schematic form of the embodiment shown inFIG. 1A (or FIG. 1C), although any other embodiments according to thepresent invention can be equivalently utilized. In the embodiment ofFIG. 2A, angled-dual-axis confocal scanning head 201 is attached to andfurther enclosed in movable carriage 202, with optical fibers 103, 104extending to the outside of movable carriage 202. A first transparentwindow 203 is mounted on movable carriage 202 for passage ofillumination beam 117 and observation beam 118. Movable carriage 202 canmove up and down in a vertical direction as defined by arrow 204,causing angled-dual-axis confocal scanning head 201 to move toward oraway from object 120 in the process. By doing so, confocal overlappingvolume 121 of illumination beam 117 and observation beam 118 furtherdeepen into (or retract from) the interior of object 120, whereby asuccession of arc-line scans that progressively deepen into object 120along a vertical cross-section plane 210 is produced, as illustrated bycurves 205, providing a vertical cross-section scan. The motion ofmovable carriage 202 can be driven by a variety of means, for instance,by coupling it to a motor (not shown in FIG. 2A) that is activated by amagnetic, hydraulic, piezoelectric, or other actuator. A skilled artisancan accordingly implement a movable stage suitable for a givenapplication.

As illumination beam 117 and observation beam 118 deepen into theinterior of object 120 in the course of vertical scanning, the change intheir respective optical path lengths becomes increasingly large, whichmay cause their respective focal volumes to no longer intersect in anoptimal manner, or even not to intersect at all at the point where thetwo beams physically meet. Furthermore, in interferometry applicationssuch as optical coherence microscopy, the optical path lengths ofillumination beam 117 and observation beam 118 must stay substantiallyfixed in order to ensure coherent interference of predominantlysingle-scattered light. To maintain the optical path lengths ofillumination beam 117 and observation beam 118 during vertical scanning,the space between movable carriage 202 and object 120 can be filled witha substantially transparent fluid 206 having an index of refraction thatis substantially the same as the index of refraction of object 120, suchthat the optical path lengths of illumination beam 117 and observationbeam 118 remain unchanged in the course of vertical scanning. The use ofoptical fibers further provides the necessary flexibility that enablesthe whole assembly of angled-dual-axis confocal scanning head 201 andmovable carriage 202 to move up and down without incurring additionalchange in the optical path lengths of illumination beam 117 andobservation beam 118. In the embodiment of FIG. 2A, movable carriage202, along with angled-dual-axis confocal scanning head 201, is disposedwithin a container 207 filled with fluid 206. An O-ring seal 211 isprovided to seal fluid 206 inside container 207, while still permittingmovable carriage 202 to move up and down relative to container 207.Container 207 is equipped with a second transparent window 208, inoptical alignment with first transparent window 203 for passage ofillumination and observation beams 117, 118. Container 207 is furtherconnected to a fluid injection system 209, serving as a reservoir forreplenishing additional fluid or receiving excess fluid as movablecarriage 202 is moving towards or away from object 120. For imaging ofhuman tissue and other biological samples, fluid 206 can be water, whichhas an index of refraction closely matching that of tissue andbiological samples.

It should be noted that certain aberrations of the illumination andobservation beams may occur as a result of successive passages of thebeams through first and second transparent windows 203, 208, fluid 206,and object 120 in the above embodiment, which may require specificdesigns of the illumination and observation focusing elements that arecorrected for these aberrations. Alternatively, auxiliary opticalelements that are properly designed for correcting such aberrations maybe implemented in the angled-dual-axis focusing means. In most cases ofa converging beam passing through a window or into another object at aninclined angle, the primary aberrations to be corrected for will bespherical aberration, astigmatism, and coma. The magnitude of theseaberrations depend upon many factors, and typically increases with NA ofthe focusing elements, the index of refraction and the thickness of thewindow, and the angle of incidence. The design of suchaberration-corrected focusing elements, or the auxiliary opticalelements for correcting aberrations, can be accomplished by a lensdesigner of ordinary skill and with the help of an optical designcomputer program such as Zemax™.

In applications where the NA of the focusing elements in theangled-dual-axis focusing means are sufficiently low and the thicknessesof windows, fluid and object through which the illumination andobservation beams successively traverse are not large, the aberrationswould be small and may not need to be corrected. In such cases, theembodiment shown in FIG. 2A can be utilized, which may incorporateadditional remedies for further minimizing aberrations. Such remediesinclude, for example, using windows made of Teflon AF™ or othermaterials that are transparent and have an index of refraction closelymatching that of water.

FIG. 2B depicts a second embodiment of an angled-dual-axis confocalscanning microscope of the present invention, pertaining to applicationswhere the aforementioned aberrations may not be negligible. Inangled-dual-axis confocal scanning microscope 250, a window assemblycomprising two flat transparent windows 254, 255 in an angledarrangement is implemented to replace single flat window 203 in FIG. 2A.The remainder of angled-dual-axis confocal scanning microscope 250shares the same components as angled-dual-axis confocal scanningmicroscope 200 shown in FIG. 2A. The window assembly is designed suchthat illumination axis 97 along with illumination beam 117 andobservation axis 96 along with observation beam 118 (see FIG. 1B) aresubstantially perpendicular to windows 255, 254, respectively. As such,the window assembly can greatly reduce coma and astigmatism that wouldotherwise be associated with using a single flat window (such as window203 in FIG. 2A). Although spherical aberrations still need to becorrected for in this case, the techniques for making such correctionsare well known in the art of lens design. For instance, a skilledartisan can make use of the design of microscope objectives that arecorrected for glass coverslips of a certain thickness to accomplish thistask.

All in all, angled-dual-axis confocal scanning microscope 200 or 250 ofthe present invention is designed such that it provides a verticalcross-section scan of an object with enhanced axial resolution, fasterspeed, and larger transverse field of view. Moreover, by movingangled-dual-axis confocal scanning microscope 200 or 250, or translatingthe object, in a transverse direction perpendicular to verticalcross-section plane 210 illustrated in FIG. 2A or FIG. 2B, a series ofvertical cross-section scans can be taken in a layer-by-layer fashion,which can be assembled to provide a three-dimensional volume image ofthe object.

FIG. 2C depicts a third embodiment of an angled-dual-axis confocalscanning microscope of the present invention. Angled-dual-axis confocalmicroscope 700, by way of example, comprises an angled-dual-axisconfocal head (ADACH) 701, a vertical translation means in the form of amovable carriage 702, and a transverse scanning means in the form oftransverse stage 212. For the purpose of illustration, angled-dual-axisconfocal scanning head 701 is in a simplified schematic form of theembodiment shown in FIG. 1A (or FIG. 1C), although any other embodimentsaccording to the present invention can be equivalently utilized. In theembodiment of FIG. 2C, angled-dual-axis confocal head 701 is attached toand further enclosed in movable carriage 702, with first and secondoptical fibers 103, 104 extending to the outside of movable carriage702. A first transparent window 703 is mounted on movable carriage 702for passage of illumination beam 117 and observation beam 118. Driven bya motor 705, movable carriage 202 can move up and down along a vertical(or axial) direction as defined by arrow 704, causing angled-dual-axisconfocal head 701 to move toward or away from object 120 in the process.By doing so, confocal overlapping volume 121 of illumination beam 117and observation beam 118 deepens into (or retracts from) the interior ofobject 120, whereby producing a vertical scan. Motor 705 can be actuatedby a variety of means, such as magnetic, hydraulic, piezoelectric, andother actuators. A skilled artisan can accordingly devise a movablecarriage mechanically driven by a motor suitable for a givenapplication.

As in the embodiment of FIG. 2A or FIG. 2B, movable carriage 702, alongwith angled-dual-axis confocal head 701, is disposed within a container707 filled with a substantially transparent fluid 706 having an index ofrefraction that is substantially the same as the index of refraction ofobject 120, such that the optical path lengths of illumination beam 117and observation beam 118 remain substantially unchanged in the course ofvertical scanning. The use of optical fibers further provides thenecessary flexibility that enables the whole assembly ofangled-dual-axis confocal head 701 and movable carriage 702 to move upand down without incurring additional changes in the optical pathlengths of illumination beam 117 and observation beam 118. An O-ringseal 709 is provided to seal fluid 706 inside container 707, while stillpermitting movable carriage 702 to move up and down relative tocontainer 707. A second transparent window 708 is mounted on container707, such that it is in optical communication with first transparentwindow 703 for passage of illumination and observation beams 117, 118.Container 707 is further connected to a fluid injection system 710,serving as a reservoir for replenishing additional fluid or receivingexcess fluid as movable carriage 702 is moving towards or away fromobject 120. As described in FIG. 2A, fluid 706 can be water, for it hasan index of refraction closely matching that of human tissue and otherbiological samples.

It should be noted that a window assembly, similar to the windowassembly shown in FIG. 2B, can be implemented to replace window 703 inapplications where aberrations need to be minimized, as described above.Further minimization of possible aberrations can be accomplished byfilling a space between second and third windows 708, 713 with a fluid,which has an index of refraction substantially close to that of fluid706. Alternatively, distance 715 can be made very small, and second andthird windows 708, 713 can be made of Teflon AF™, to further decreasecoma and astigmatism. Although spherical aberrations still need to becorrected for in this case, the techniques for making such correctionsare well known in the art of lens design. For instance, a skilledartisan can make use of the design of microscope objectives that arecorrected for glass coverslips of a certain thickness to accomplish thistask.

Angled-dual-axis confocal scanning microscope 700 further comprises atransverse stage 712 for producing transverse scans. A third transparentwindow 713, in optical communication with second transparent window 708,is mounted on transverse stage 712 for passage of illumination andobservation beams 117, 118. Object 120 is in turn placed on thirdtransparent window 713. Transverse stage 712 is mechanically coupled totwo or more ball bearings (or wheels) 714, which enable transverse stage212 along with object 120 to translate relative to angled-dual-axisconfocal head 701 along transverse directions perpendicular to thevertical direction 704, whereby producing a transverse scan. Ballbearings 714 also serve to keep the distance 715 between secondtransparent window 708 and third transparent window 713 constant, so asto preserve the optical path lengths of illumination and observationbeams 117, 118 in the course of scanning.

As such, angled-dual-axis confocal scanning microscope 700 of thepresent invention is capable of providing transverse and vertical scansin various ways. For example, it can produce a line scan along thevertical direction 704, termed a vertical-line scan hereinafter; avertical cross-section scan comprising a plurality of vertical-linescans that are assembled along a transverse direction perpendicular tothe vertical direction 704; a line scan along a transverse directionperpendicular to the vertical direction 704, termed a transverse-linescan hereinafter; and a transverse cross-section scan comprising aplurality of transverse-line scans assembled in a transverse planeperpendicular to the vertical direction 704; and so on. Furthermore, byassembling a plurality of transverse cross-section scans thatprogressively deepens into the object, by assembling a plurality ofvertical cross-section scans that move incrementally in a transversedirection (perpendicular to each vertical cross-section scan), or byassembling an assortment of transverse-line scans along differenttransverse directions and vertical-line scans, a three-dimensionalvolume image of the object can be constructed.

For tissue imaging applications, the wavelength of light generallyranges from about 0.8 microns to 1.6 microns, since biological tissuesamples are particularly transparent in this range. Embodiments of theangled-dual-axis confocal scanning microscope of the present inventionare capable of achieving a resolution of about 1-5 microns in the axial(e.g., the vertical direction shown in FIGS. 2A-2C) as well as thetransverse directions, by use of illumination and observation lenseswith NA typically ranging from 0.1 to 0.4, and the intersecting angle θtypically ranging from 45° to 90°. The vertical cross-section scan areais typically on the order of about 0.5-1 millimeter in both directions.In terms of scanning capabilities, the fast scan rate along an arc-linetypically ranges from 1 to 10 KHz, and the maximum rotation angle (e.g.,φ in FIG. 1A) from a neutral position of the scanning mirror (e.g.,scanning mirror 111 in FIG. 1A) may range from one to several degrees.Generally, the smaller and the lighter the scanning mirror, the fasterthe scanning rate. For instance, using a silicon micro-machined scanningmirror can provide scanning rates greater than 10 kHz. The verticalscanning can be performed at a slower rate of 10-60 Hz, which definesthe frame rate of vertical cross-section scanning and is in the range ofvideo-rate scanning. Transverse stage 712 scans at a rate slower thanthe arc-line scanning rate, typically in the range of 0.1 Hz to 100 Hz.

The specific numbers provided above are designed for tissue imaging, toillustrate the utility and the performance of the present invention as away of example. A skilled artisan can utilize the model calculationprovided in Ser. No. 09/628,118 to design an angled-dual axis confocalscanning microscope in accordance with the present invention, for agiven application.

It should be pointed out that although optical fibers, particularlysingle-mode fibers, are preferable as optical coupling means amongvarious optical elements in this invention, and are used throughout thisspecification wherever optical coupling is called for, other suitableoptical coupling means can be alternatively implemented in variousangled-dual-axis optical coherence scanning systems of this invention,without deviating from the principle and the scope of the presentinvention.

FIG. 3A depicts a first embodiment of an angled-dual-axis opticalcoherence scanning microscope of the present invention. Angled-dual-axisoptical coherence microscope 300 comprises an angled-dual-axis confocalscanning microscope (ADACSM) 301, a beam-splitting means preferably inthe form of a fiber-based beamsplitter 302, a light source 303 having ashort coherence length, and a reference optical fiber 304. By way ofexample, angled-dual-axis confocal scanning microscope 301 is in theform of one of the embodiments shown in FIGS. 2A-2C, although otherembodiments in accordance with the present invention can also beimplemented. Beamsplitter 302 is optically coupled to light source 303and angled-dual-axis confocal scanning microscope 301 in such a way thatit diverts a portion of an output beam emitted from light source 303 tofirst optical fiber 103 of angled-dual-axis confocal scanning microscope301, providing an illumination beam 117, and a remainder of the outputbeam to reference optical fiber 304, providing a reference beam. Anobservation beam 118 collected by angled-dual-axis confocal scanningmicroscope 301 from confocal overlapping volume 121 within object 120 isdelivered by way of second optical fiber 104. Second optical fiber 104and reference fiber 304 are joined by a fiber-optic coupler 305, andoptical path lengths of first, second and reference fibers 103, 104, 304are so selected to ensure coherent interference upon combining thereference and observation beams at fiber-optic coupler 305. The twooutputs of fiber-optic coupler 305 are in turn fed to two opticaldetectors 309, 310, such that a balanced detection scheme is utilizedfor optimizing the signal-to-noise of detection. The underlyingprinciple of the balanced detection technique as well as its advantagesin fiber-optic interferometers are well known in the art, as describedby Rollins et al. in “Optimal interferometer designs for opticalcoherence tomography”, Optics Letters, 24(21), pp. 1484 (1999), and byPodoleanu in “Unbalanced versus balanced operation in an opticalcoherence tomography system”, Applied Optics, 39(1), pp. 173 (2000),incorporated herein by reference.

Angled-dual-axis optical coherence scanning microscope 300 furthercomprises a frequency shifting means 306 optically coupled to, by way ofexample, second optical fiber 104 of angled-dual-axis confocal scanningmicroscope 301, such that the frequency of the observation beam isshifted relative to the frequency of the reference beam. Alternatively,a frequency shifting means may be optically coupled to first opticalfiber 103, which also results in the frequency of the observation beambeing shifted relative to the frequency of the reference beam. Moreover,a frequency shifting means can be coupled to reference optical fiber304, such that the frequency of the reference beam is shifted relativeto the frequency of the observation beam. The end result in each case isthat coherent interference between the reference and observation beamsis modulated at a heterodyne beat frequency given by the relativefrequency shift between the two beams, allowing for heterodyne balanceddetection. In addition, an adjustable optical delay device 307 iscoupled to, by way of example, second optical fiber 104, so as tomaintain coherent interference between the reference and observationbeams at fiber-optic coupler 305 where they are combined. An opticaldelay device may be alternatively coupled to reference fiber 304, orfirst optical fiber 103, for the same purpose. In applications wherelight source 303 has a short coherence length, optical delay device 307can be adjusted such that mostly single-scattered light in observationbeam 118 is coherent with the reference beam at fiber-optic coupler 305and multiple-scattered light, which traverses over a larger optical pathlength in observation beam 118, does not contribute to the coherentinterference, therefore providing further filtering ofmultiple-scattered light upon detection. To enhance the signal-to-noiseratio of detection of weak optical signals, an optical amplifier 308,such as a two-port fiber amplifier or a semiconductor optical amplifier,is optically coupled to second optical fiber 104, so as to boost up thepower of the observation beam. An amplified observation beam has anadditional advantage of rendering faster scanning rates and consequentlyhigher pixel rates without appreciable loss in the signal-to-noiseratio, because a shorter integration time per pixel of an image isrequired upon data collection. The balanced detection scheme allowssubtraction of amplifier noise, since preponderance of spontaneousemission of optical amplifier 308 would not occur at the heterodyne beatfrequency described above.

In general, light source 303 in the above embodiment can be an opticalfiber amplifier, a semiconductor optical amplifier, a fiber laser, asemiconductor laser, a diode-pumped solid state laser, or a broadbandOCT source commonly used in optical coherence tomography applications.It may be polarized, or unpolarized. The light source preferably has acoherence length of less than 3000 microns (for applications involvingimaging within a highly scattering medium such as human skin,wavelengths in the range of 0.8 to 1.6 microns typically allow imagingto depths of no more than 3,000 microns). For biological applications,the light source should produce light in the wavelength range of 0.8 to1.6 microns, since biological tissues are particularly transparent inthis range. Beamsplitter 302 can be a fiber-optic coupler, such as anevanescent wave coupler or a fused fiber coupler. Various opticalfibers, such as first, second, and reference optical fibers 103, 104,304, are preferably single-mode fibers, for single-mode fibers have theadvantage of simplicity and automatic assurance of the mutual spatialcoherence of the observation and reference beams upon mixing anddetection.

In one case where light source 303 in the above embodiment producespolarized light and beamsplitter 302 is a polarizing beamsplitter, theorientation of polarizing beamsplitter 302 relative to the polarizationof the light can be used to control the ratio of optical power betweenthe illumination and reference beams. First, second, and referenceoptical fibers 103, 104, 304 are preferably polarization maintaining(PM) fibers, to provide control of the polarizations of theillumination, observation and reference beams throughout the system. Inthis case, the reference and observation beams can be brought into thesame polarization by an appropriate rotation of second optical fiber 104or reference optical fiber 304, before they are joined at fiber-opticcoupler 305. Alternatively, a polarization rotation means, such as aFaraday rotator or a rotatable fiber connector, can be coupled to eitherreference optical fiber 304 or second optical fiber 104, such that thereference and observation beams have substantially the same polarizationwhen combined.

In another case where light source 303 is a polarized light source,beamsplitter 302 may alternatively be a polarization maintainingfiber-optic coupler. Likewise, polarization maintaining fibers should beimplemented throughout the system, to provide control of thepolarization states of the beams, thereby ensuring maximum coherentinterference between the observation and reference beams upon beingcombined.

The embodiment described above can be further used to provide specificinformation pertaining to the polarization state of light upon beingreflected from a polarization-altering, such as birefrigent-scattering,medium. Many biological tissues, such as tendons, muscle, nerve, bone,cartilage and teeth, exhibit birefrigence due to their linear or fibrousstructure. Birefrigence causes the polarization state of light to bealtered (e.g., rotated) in a prescribed manner upon refection. Skin isanother birefrigent medium. Collagen contained in skin is a weaklybirefrigent material. Moreover, at temperatures between 56-65° C.,collagen denatures and loses its birefrigence. Thus, by detectinginduced changes in the polarization state of light reflected from a skinsample, an image representing the regions of skin where thermal injuryoccurs can be identified. FIG. 3B depicts a second embodiment of anangled-dual-axis optical coherence scanning microscope of the presentinvention, pertaining to applications where polarized light is used toprobe a birefrigent-scattering (or other polarization-altering) medium.By way of example, angled-dual-axis optical coherence scanningmicroscope 350 comprises an angled-dual-axis confocal scanningmicroscope (ADACSM) 351, a polarizing beamsplitter 352, a polarizedlight source 353, and a reference polarization maintaining (PM) opticalfiber 354. As in FIG. 3A, angled-dual-axis confocal scanning microscope351 is in the form of one of the embodiments shown in FIGS. 2A-2C, withfirst and second optical fibers 103, 104 being polarization maintaining(PM) fibers capable of supporting two orthogonal polarizations.Polarizing beamsplitter 352 is optically coupled to polarized lightsource 353 by way of a third PM fiber 359, such that it diverts aportion of a polarized output beam emitted from light source 353 tofirst PM fiber 103, providing an illumination beam 117 withP-polarization to angled-dual-axis confocal scanning microscope 351, anda remainder of the output beam to reference PM fiber 354, providing areference beam with S-polarization. P-polarization and S-polarizationare orthogonal to each other. The orientation of polarizing beamsplitter352 relative to the polarization of the output beam from light source353 can be used to control the ratio of optical power between theillumination and reference beams. An observation beam 118 reflected fromconfocal overlapping volume 121 within an object 120 carries bothP-polarization and S-polarization, where the presence of S-polarizationis resulted from birefrigent (or other polarization-altering)“scatterers” in object 120. A frequency shifting means 356 is opticallycoupled to second PM fiber 104, such that the frequency of observationbeam 118 is shifted relative to the frequency of the reference beam.Second PM fiber 104 and reference PM fiber 354 are joined by apolarization maintaining (PM) fiber-optic coupler 355, where onlyobservation beam 118 with S-polarization is coherently combined with thereference beam that has only S-polarization. The two outputs offiber-optic coupler 355 are in turn fed to two optical detectors 360,361, so as to utilize balanced detection scheme for optimizing thesignal-to-noise of detection. The amplitude of resulting heterodyne beatfrequency signal, i.e, S-polarized Signal depicted in FIG. 3B,corresponds only to the amplitude of reflectance of light withS-polarization, from which an image representing birefrigent (and/orother polarization-altering) “scatterers” in object 120 can beconstructed. Take skin as an example. If thermal damage occurs to a skinsample, the amplitude of S-polarized Signal depicted would be reducedcompared to that corresponding to normal skin.

In angled-dual-axis optical coherence scanning microscope 350, afrequency shifting means may be alternatively coupled to reference PMfiber 354, such that the frequency of the reference beam is shiftedrelative to the frequency of the observation beam. A frequency shiftingmeans may be also coupled to first optical fiber 103, for shifting thefrequency of illumination beam 117 and hence observation beam 118relative to the frequency of the reference beam. In each case the endresult is that coherent interference between the reference andobservation beams is modulated at a heterodyne beat frequency given bythe relative frequency shift between the two beams, allowing forheterodyne balanced detection. In addition, an adjustable optical delaydevice 357 is coupled to, by way of example, second PM fiber 104, so asto maintain coherent interference between the reference and observationbeams at PM fiber-optic coupler 355 where they are combined. An opticaldelay device may be alternatively coupled to reference PM fiber 354, orfirst optical fiber 103. In each case, the optical delay device can alsobe adjusted to provide further filtering of multiple-scattered lightupon detection, as described above. To enhance the signal-to-noise ratioupon detection, an optical amplifier 358, such as a two-port fiberamplifier or semiconductor optical amplifier, is coupled to second PMfiber 104, so as to boost up the power of the observation beam. Anamplified observation beam brings an additional advantage of allowingfaster scanning rates and consequently higher pixel rates withoutappreciable loss in the signal-to-noise ratio, because a shorterintegration time per pixel of an image is required upon data collection.The use of balanced detection in this case allows subtraction ofamplifier noise, since spontaneous emission of optical amplifier 358would not occur at the heterodyne beat frequency described above.

It should be noted that the aforementioned embodiment can also be usedto detect P-polarization of the observation beam, if so desired in agiven application. In such a case, a polarization rotation means, suchas a rotatable fiber connector or a Faraday rotator, can be coupled toreference PM fiber 354, serving to rotate the polarization of thereference beam by 90-degree and hence rendering a reference beam withP-polarization. Upon combining the reference and observation beams at PMfiber-optic coupler 355, only the observation beam with P-polarizationinterferes coherently with the reference beam that now has onlyP-polarization. As such, the amplitude of resulting heterodyne beatfrequency signal measures only the amplitude of reflectance of lightwith P-polarization.

The second embodiment shown in FIG. 3B can be further modified to allowboth P-polarization and S-polarization of the observation beam to bedetected, hence providing enhanced contrast of an image pertaining tobirefrigent-scattering (and/or other polarization-altering) “scatterers”in an object. FIG. 3C shows a third embodiment of an angled-dual-axisoptical coherence scanning microscope of the present invention.Angled-dual-axis optical coherence scanning microscope 370, by way ofexample, comprises an angled-dual-axis confocal scanning microscope(ADACSM) 371, a first polarizing beamsplitter 372, a polarized lightsource 373, and a reference polarization maintaining (PM) optical fiber374. As in FIG. 3B, angled-dual-axis confocal scanning microscope 371 isin the form of one of the embodiments shown in FIGS. 2A-2C, with firstand second optical fibers 103, 104 being polarization maintaining (PM)fibers capable of supporting two orthogonal polarizations. Polarizingbeamsplitter 372 is optically coupled to polarized light source 373 byway of a third PM fiber 375, such that it diverts a portion of apolarized output beam emitted from light source 373 to first PM fiber103, providing an illumination beam 117 with P-polarization toangled-dual-axis confocal scanning microscope 371, and a remainder ofthe output beam to reference PM fiber 374, providing a reference beamwith S-polarization. P-polarization and S-polarization are mutuallyorthogonal. The orientation of polarizing beamsplitter 372 relative tothe polarization of the output beam from light source 373 can be used tocontrol the ratio of optical power between the illumination andreference beams. An observation beam 118 reflected from confocaloverlapping volume 121 within an object 120 carries both P-polarizationand S-polarization, owing to birefrigent (or otherpolarization-altering) “scatterers” in object 120. A frequency shiftingmeans 376 is optically coupled to first PM fiber 103, such that thefrequency of illumination beam 117 and hence the frequency ofobservation beam 118 are shifted relative to the reference beam. SecondPM fiber 104 is optically coupled to a second polarizing beamsplitter377, which routes S-polarization and P-polarization of observation beam118 to forth PM fiber 378 and fifth PM fiber 379, respectively.Reference PM fiber 374 is optically coupled to a polarization rotationmeans 380, such as a rotatable fiber-optic connector or a Faradayrotator, serving to rotate the polarization of the reference beam by45-degree, effectively rendering the reference beam with bothP-polarization and S-polarization. A third polarizing beamsplitter 381in turn receives the reference beam from polarization rotation means380, and routes S-polarization and P-polarization of the reference beamto sixth PM fiber 382 and seventh PM fiber 383, respectively. Fourth PMfiber 378 and sixth PM fiber 382 are joined by a first polarizationmaintaining (PM) fiber-optic coupler 384, where S-polarization of theobservation beam is coherently combined with S-polarization of thereference beam. Illustrated as S-polarized Signal in FIG. 3C, theamplitude of resulting heterodyne beat frequency signal provides ameasure of the amplitude of reflectance of light with S-polarization.The two outputs of first polarization maintaining (PM) fiber-opticcoupler 384 are fed to first and second optical detectors 385, 386, suchthat balanced heterodyne detection scheme is utilized for optimizing thesignal-to-noise ratio of detection. Likewise, fifth PM fiber 379 andseventh PM fiber 383 are joined by a second polarization maintaining(PM) fiber-optic coupler 387, where P-polarization of the observationbeam is coherently combined with P-polarization of the reference beam.Illustrated as P-Polarized Signal in FIG. 3C, the amplitude of resultingheterodyne beat frequency signal measures only the amplitude ofreflectance of light with P-polarization. The two outputs of secondpolarization maintaining (PM) fiber-optic coupler 387 are fed to thirdand fourth optical detectors 388, 389, to provide for a balancedheterodyne detection scheme. The simultaneous detection of bothP-polarization and S-polarization of the observation beam yields anenhanced contrast image representing birefrigent (and/or otherpolarization-altering) “scatterers” in object 120. For example,P-Polarized Signal and S-Polarized Signal can be combined in variousways to produce a polarization image, such as [(P-PolarizedSignal)−(S-Polarized Signal)]/[(P-Polarized Signal)+(S-PolarizedSignal)]. This particular way of processing the signals can helpdiscriminate the portion of light reflected by the polarization-alteringscatterers from the portion of light that is randomly polarized bymultiple-scattered events.

Angled-dual-axis optical coherence scanning microscope 370 furthercomprises a first adjustable optical delay device 390 optically coupledto, by way of example, sixth PM fiber 382, to maintain coherentinterference between the S-polarized reference and observation beamsupon mixing and detection. An adjustable optical delay device may bealternatively coupled to fourth PM fiber 378, to achieve the samepurpose. Moreover, a second adjustable optical delay device 391 isoptically coupled to, by way of example, seventh PM fiber 383, tomaintain coherent interference between the P-polarized reference andobservation beams upon mixing and detection. An adjustable optical delaydevice can be alternatively coupled to fifth PM fiber 379, for the samepurpose. Separate optical delay adjustments for P-polarization andS-polarization are necessary, because the polarization mode dispersion(PDM) of the PM fibers causes the two orthogonal polarization modes totravel over different optical path lengths along a given length of thePM fiber. Therefore, these optical delay devices can be separatelyadjusted to provide coherence interference of the two beams in eachpolarization mode. In addition, an optical amplifier 392, such as atwo-port polarization-sensitive fiber optical amplifier or semiconductoroptical amplifier, is optically coupled to second PM fiber 104, to boostup the power of the observation beam. An amplified observation beamallows faster scanning rates and consequently higher pixel rates withoutappreciable loss in the signal-to-noise ratio, because a shorterintegration time per pixel of an image is required upon data collection.The balanced heterodyne detection schemes implemented for measuring bothS-polarized and P-polarized signals enable the amplifier noise to besubtracted in each case, since preponderance of spontaneous emission ofoptical amplifier 392 would not occur at the heterodyne beat frequency.

In the embodiment of FIG. 3B or FIG. 3C, various optical fibers employedare preferably single-mode, polarization maintaining fibers capable ofsupporting two orthogonal polarizations. The polarization modedispersion (PMD) in the interferometer can be minimized by use of shortsingle-mode fiber lengths in the arms of the observation and referencebeams, as a particular application warrants. If other types of opticalfibers are to be alternatively employed in the system, it would bepreferable to implement fiber-optic polarization controllers accordinglyin the arms of the interferometer, so as to provide control of thepolarizations of the observation and reference beams. The use of suchfiber-optic polarization controllers with non PM single-mode fibers iswell known in the art of optical coherence tomography.

It should also be noted that light source 353 in FIG. 3B (or lightsource 373 in FIG. 3C) can be an unpolarized light source, in general.In such a case, the use of polarizing beamsplitter 352 (or polarizingbeamsplitter 372 in FIG. 3C) would generate a polarized illuminationbeam and a polarized reference beam with orthogonal polarizations froman unpolarized beam emitted from the light source. A disadvantage ofusing an unpolarized light source is, however, that the ratio of opticalpower between the illumination and reference beams cannot be efficientlyadjusted to best suit a particular application.

In all cases involving polarized light, it is important to carefullycontrol the polarization states of the observation and reference beams,such that coherent interference between the two beams is maximized whencombined. Those skilled in the art of fiber-optic interferometers orgyroscopes will know how to implement various polarization maintainingand/or controlling means to achieve this purpose.

FIG. 4 shows a fourth embodiment of an angled-dual-axis opticalcoherence scanning microscope of the present invention. Angled-dual-axisoptical coherence scanning microscope 400 comprises an angled-dual-axisconfocal scanning microscope (ADACSM) 401, a light source 402, and areference optical fiber 403. A first output port 404 of light source 402is optically coupled to first optical fiber 103, transmitting anillumination beam 117 to angled-dual-axis confocal scanning microscope401. A second output port 405 of light source 402 is optically coupledto reference optical fiber 403, providing a reference beam. Anobservation beam 118 collected by angled-dual-axis confocal scanningmicroscope 401 is delivered by way of second optical fiber 104 ofangled-dual-axis confocal scanning microscope 401. Reference opticalfiber 403 and second optical fiber 104 are further joined by afiber-optic coupler 406, and optical path lengths of first, second andreference fibers 103, 104, 403, are so selected to ensure coherentinterference upon combining the reference and observation beams atfiber-optic coupler 406. The two outputs of fiber-optic coupler 406 arefed to two optical detectors 410, 411, such that a balanced detectionscheme is utilized for maximizing the signal-to-noise ratio ofdetection.

Angled-dual-axis optical coherence scanning microscope 400 furthercomprises a frequency shifting means 407 optically coupled, by way ofexample, second optical fiber 104, such that the frequency ofobservation beam 118 is shifted relative to the frequency of thereference beam. A frequency shifting means can also be coupled to firstoptical fiber 103, for the same purpose of shifting the frequency of theobservation beam. Alternatively, a frequency shifting means may beoptically coupled to reference optical fiber 403, such that thefrequency of the reference beam is shifted relative to the frequency ofthe observation beam. The end result in each case is that coherentinterference between the reference and observation beams is modulated ata heterodyne beat frequency given by the relative frequency shiftbetween the two beams, allowing for heterodyne balanced detection. Inaddition, an adjustable optical delay device 408 is optically coupledto, by way of example, second optical fiber 104, so as to maintaincoherent interference between the reference and observation beams atfiber-optic coupler 406 where they are combined. An optical delay devicemay be alternatively coupled to reference optical fiber 403, or firstoptical fiber 103. In each case, the optical delay device can also beadjusted to provide further filtering of multiple-scattered light upondetection, as described above. To enhance the signal-to-noise ratio ofdetection of weak optical signals, an optical amplifier 409, such as atwo-port fiber amplifier or a semiconductor optical amplifier, isoptically coupled to second optical fiber 104, so as to boost up thepower of the observation beam. An amplified observation beam allowsfaster scanning rates and consequently higher pixel rates withoutappreciable loss in the signal-to-noise ratio, because a shorterintegration time per pixel of an image is required upon data collection.The balanced detection scheme allows subtraction of amplifier noise,since preponderance of spontaneous emission of optical amplifier 409would not occur at the heterodyne beat frequency described above.

Light source 402 in the above embodiment is preferably a short coherencelength source, such as an OCT source commonly used for OCT applicationsthat is modified to have two output-ports. The light source can also bean optical fiber amplifier, a semiconductor optical amplifier, a fiberlaser, a semiconductor laser, or a diode-pumped solid state laser,equipped with dual output-ports. The light source preferably has acoherence length of less than 3000 microns. For biological applications,the light source should produce light in the wavelength range of 0.8 to1.6 microns, since biological tissues are particularly transparent inthis range. Various optical fibers, such as first, second, and referenceoptical fibers 103, 104, 403, are preferably single-mode fibers, forsingle-mode fibers have the advantage of simplicity and automaticassurance of the mutual spatial coherence of the observation andreference beams upon mixing and detection. If polarized light isprovided by light source 402, first, second, and reference opticalfibers 103, 104, 403, are preferably polarization maintaining fibers. Insuch a case, the reference and observation beams can be brought into thesame polarization by an appropriate rotation of either second opticalfiber 104 or reference optical fiber 403, before they are joined byfiber-optic coupler 406. Alternatively, a polarization rotation means,such as a Faraday rotator or a rotatable fiber connector, can beoptically coupled to either reference optical fiber 403 or secondoptical fiber 104, such that the reference and observation beams havesubstantially the same polarization when combined.

Moreover, the embodiment of FIG. 4 can be further modified, in wayssimilar to the embodiments depicted in FIGS. 3B-3C, to image apolarization-altering (e.g., a birefrigent-scattering) medium. Thoseskilled in the art can implement such modifications in accordance withthe present invention for a given application.

It should be noted that to provide the frequency shifting means, a phasemodulator can be implemented in the embodiments describe above formodulating the phase of either the reference or observation beam, suchthat heterodyne coherent interference is produced between the referenceand observation beams. The phase modulator can be a piezoelectric fiberstretcher, an electro-optic crystal, an acousto-optic modulator, or anyother phase modulator known in the art. It will be apparent to oneskilled in the art of heterodyne interferometry techniques that thereexist many ways of modulating the phase or shifting the frequency of theobservation beam (or the reference beam), such that a detectable beatfrequency is generated for detection.

As described above, the angled-dual-axis arrangement between theillumination and observation beams in an angled-dual-axis confocalscanning microscope of the present invention has an inherent advantageof filtering out multiple-scattered light in the illumination beam. Andmultiple-scattered light can be further eliminated by employing low NAfocusing elements (or lenses) in the angled-dual-axis confocal scanningmicroscope. In an angled-dual-axis optical coherence scanning microscopeof the present invention, as exemplified by the embodiments depicted inFIGS. 3A-3C and FIG. 4, multiple-scattered light that originates withinthe observation beam can be additionally minimized, since themultiple-scattered light in the observation beam are not coherent withthe reference beam, owing to the fact that multiple-scattered lighttravels over a longer optical path length than single-scattered lightupon mixing with the reference beam. As such, an angled-dual-axisoptical coherence scanning microscope of the present invention canachieve much higher sensitivity and larger dynamic range in detection,and hence enhanced contrast when imaging within a scattering medium.This capability is particularly desirable when imaging in a highlyscattering medium, such as biological tissue.

All in all, the unique design of the angled-dual-axis optical coherencescanning microscope of the present invention enables it to achieve manyadvantages concurrently, as stated throughout this specification, incontrast to many prior art systems where an improvement in one propertyoften occurs at the expense of adversely affecting another property.

When using an angled-dual-axis optical coherence system to scan anobject in order to create an image, the beat frequency resulting fromheterodyne interference of the reference and observation beams should beselected with consideration of the scanning rate carried out theangled-dual-axis confocal scanning microscope in the system. Morespecifically, the beat frequency should be substantially higher than therate at which pixels inside the object are measured, such that theinterference magnitude is measured over several cycles. The highestpossible resolution of the image corresponds to the dimensions of theconfocal overlapping volume, and proper sampling of a region to beimaged requires that more than two pixels per image point be obtained.For example, if pixels are measured at a rate of 1 MHz (fast enough forvideo), then the beat frequency should be at least about 10 MHz. Thiswill provide 10 interference fringes per pixel, which is sufficient toprovide an accurate measure of the reflectance of each pixel. The pixelscanning speed and the beat frequency may depend upon the particularapplication, of course. If high accuracy is required of the reflectancemeasurements, then the beat frequency may be increased or the pixelscanning rate may be reduced. Those skilled in the art can accordinglydevise a working relationship between the pixel scanning rate and thecorresponding beat frequency for a given application.

It should be understood that the embodiments shown FIGS. 3A-3C and FIG.4 provide only a few of many angled-dual-axis optical coherence scanningmicroscopes in accordance with the present invention. A skilled artisanmay alter those exemplary embodiments in various ways, so as to bestsuit practical applications, without deviating from the principle andthe scope of the present invention.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions, andalterations can be made herein without departing from the principle andthe scope of the invention. Accordingly, the scope of the presentinvention should be determined by the following claims and their legalequivalents.

What is claimed is:
 1. An apparatus for performing angled-dual-axisoptical coherence scanning microscopy within a sample, comprising: a) alight-generating means for generating an illumination beam and areference beam; b) an angled-dual-axis confocal scanning microscope,comprising: i) a first optical fiber having first and second ends, fromsaid first end said illumination beam emerges; ii) a second opticalfiber having first and second ends; iii) an angled-dual-axis focusingmeans for focusing said illumination beam to an illumination focalvolume along an illumination axis within said sample and for receivingan observation beam emanated from an observation focal volume along anobservation axis within said sample such that said observation beam isfocused onto said first end of said second optical fiber; and iv) ascanning means;  wherein said illumination axis and said observationaxis intersect at an angle within said sample, such that saidillumination focal volume and said observation focal volume intersect ata confocal overlapping volume, and wherein said scanning means iscapable of directing said illumination and observation beams in such away that said illumination axis and said observation axis remainintersecting at said angle and that said confocal overlapping volumemoves within said sample; and c) a beam-combining means for combiningsaid reference beam and said observation beam, such that coherentinterference between said reference beam and said observation beam isproduced.
 2. The apparatus of claim 1 wherein said light-generatingmeans comprises: a) a light source for emitting an output beam; and b) abeam-splitting means in optical communication with said light source andsaid angled-dual-axis confocal scanning microscope, such that a portionof said output beam is passed into said second end of said first opticalfiber and a remainder of said output beam is routed into a first end ofa reference optical fiber, wherein said portion of said output beamconstitutes said illumination beam and said remainder of said outputbeam serves as said reference beam.
 3. The apparatus of claim 2 whereinsaid light source comprises an element selected from the groupconsisting of optical fiber amplifiers, fiber lasers, semiconductoroptical amplifiers, semiconductor lasers, and diode-pumped solid statelasers, and broadband OCT light sources.
 4. The apparatus of claim 2wherein said beam-splitting means comprises an element selected from thegroup consisting of fiber-optic couplers, polarization maintainingfiber-optic couplers, beamsplitters, and polarizing beamsplitters. 5.The apparatus of claim 2 wherein said beam-combining means comprises afiber-optic coupler, wherein said reference optical fiber and saidsecond optical fiber are joined by said fiber-optic coupler.
 6. Theapparatus of claim 5 wherein said fiber-optic coupler is a polarizationmaintaining fiber-optic coupler.
 7. The apparatus of claim 1 whereinsaid light-generating means comprises a light source having first andsecond output-ports, wherein said first output-port is optically coupledto said second end of said first optical fiber, transmitting saidillumination beam to said first optical fiber, and wherein said secondoutput port is optically coupled to a first end of a reference opticalfiber, providing said reference beam.
 8. The apparatus of claim 7wherein said light source comprises an element selected from the groupconsisting of optical fiber amplifiers, fiber lasers, semiconductoroptical amplifiers, semiconductor lasers, and diode-pumped solid statelasers.
 9. The apparatus of claim 7 wherein said beam-combining meanscomprises a fiber-optic coupler, wherein said reference optical fiberand said second optical fiber are joined by said fiber-optic coupler.10. The apparatus of claim 9 wherein said fiber-optic coupler is apolarization maintaining fiber-optic coupler.
 11. The apparatus of claim1 wherein said scanning means comprises an arc-line scanning meansinterposed between said angled-dual-axis focusing means and said sample,wherein said arc-line scanning means causes said confocal overlappingvolume to move across said sample in a direction substantiallyperpendicular to said illumination axis and said observation axis,thereby producing an arc-line scan.
 12. The apparatus of claim 11wherein said arc-line scanning means comprises one or more scanningelements selected from the group consisting of scanning mirrors,reflectors, acousto-optic deflectors, electro-optic deflectors,mechanical scanning mechanisms, and Micro-Electro-Mechanical-Systems(MEMS) scanning micro-mirrors.
 13. The apparatus of claim 12 whereinsaid one or more scanning elements comprise a single scanning mirror,wherein said scanning mirror is flat and can be pivoted about one axis.14. The apparatus of claim 13 wherein said scanning mirror is a siliconmicro-machined mirror.
 15. The apparatus of claim 12 wherein said one ormore scanning elements comprise two scanning mirrors that are coplanar,wherein said scanning mirrors are flat and can co-rotate about onecommon axis.
 16. The apparatus of claim 15 wherein said scanning mirrorsare silicon micro-machined mirrors.
 17. The apparatus of claim 12wherein said one or more scanning elements comprise two scanningmirrors, wherein each of said scanning mirrors is flat and can bepivoted about one or more axes, and wherein said scanning mirrors areconfigured such that they can be operated in synchronous motion.
 18. Theapparatus of claim 17 wherein said scanning mirrors are siliconmicro-machined mirrors.
 19. The apparatus of claim 1 wherein saidscanning means comprises a vertical scanning means, wherein saidvertical scanning means comprises: a) a vertical translation means; andb) a compensation means for ensuring said intersection of saidillumination focal volume and said observation focal volume; whereinsaid vertical translation means causes said angled-dual-axis focusingmeans and said first ends of first and second optical fibers to move inunity relative to said sample in a vertical direction, whereby saidconfocal overlapping volume deepens progressively into said sample,providing a vertical scan.
 20. The apparatus of claim 19 wherein saidcompensation means comprises a fluid filling a space between saidangled-dual-axis focusing means and said sample, wherein said fluid istransparent to said illumination beam and said observation beam, andwherein said fluid has an index of refraction that is the same as anindex of refraction of said sample, such that the optical path lengthsof said illumination beam and said observation beam remain unchanged inthe course of said vertical scan.
 21. The apparatus of claim 20 furthercomprising a window assembly interposed between said angled-dual-axisfocusing means and said fluid for passage of said illumination beam andsaid observation beam.
 22. The apparatus of claim 21 wherein said windowassembly comprises a transparent flat window.
 23. The apparatus of claim21 wherein said window assembly comprises first and second transparentflat windows in an angled arrangement, such that said illumination axisis perpendicular to said first flat window and said observation axis isperpendicular to said second flat window.
 24. The apparatus of claim 20further comprising a transparent window interposed between said fluidand said sample for passage of said illumination beam and saidobservation beam.
 25. The apparatus of claim 20 wherein said fluid iscontained in a sealed hydraulic system, wherein said hydraulic systemincludes a reservoir for replenishing and receiving excess fluid in thecourse of said vertical scan.
 26. The apparatus of claim 1 wherein saidscanning means comprises a transverse scanning means, wherein saidtransverse scanning means causes said angled-dual-axis focusing meansand said first ends of first and second optical fibers to move in unityrelative to said sample along transverse directions that aresubstantially perpendicular to said vertical direction, whereby saidconfocal overlapping volume moves across said sample along saidtransverse directions, producing a transverse scan.
 27. The apparatus ofclaim 1 wherein said angled-dual-axis focusing means comprises one ormore elements selected from the group consisting of refractive lenses,diffractive lenses, GRIN lenses, focusing gratings, micro-lenses,holographic optical elements, binary lenses, curved mirrors, flatmirrors, and prisms.
 28. The apparatus of claim 27 wherein said one ormore elements comprise a single refractive lens, where said refractivelens provides said illumination axis and said observation axis.
 29. Theapparatus of claim 27 wherein said one or more elements comprise anillumination focusing element and an observation focusing element,wherein said illumination focusing element provides said illuminationaxis, and wherein said observation focusing element provides saidobservation axis.
 30. The apparatus of claim 29 wherein saidillumination focusing element and said observation focusing element areof the same type, comprising a focusing element selected from the groupconsisting of refractive lenses, diffractive lenses, GRIN lenses,micro-lenses, binary lenses, and curved mirrors.
 31. The apparatus ofclaim 30 wherein said focusing element has a numerical aperture (NA) inthe range of 0.1 to 0.4.
 32. The apparatus of claim 29 furthercomprising a first collimating lens, wherein said first collimating lensreceives said illumination beam from said first end of said firstoptical fiber and passes a collimated illumination beam to saidillumination focusing element.
 33. The apparatus of claim 32 furthercomprising a second collimating lens, wherein said second collimatinglens receives said observation beam from said observation focusingelement and focuses said observation beam to said first end of saidsecond optical fiber.
 34. The apparatus of claim 29 further comprisingone or more mirrors for beam-aligning and beam-deflecting, wherein saidone or more mirrors receive said illumination beam from saidillumination focusing element and direct said illumination beam to saidillumination focal volume within said sample, and wherein said one ormore mirrors collect said observation beam emanated from saidobservation focal volume and pass said observation beam to saidobservation focusing element.
 35. The apparatus of claim 1 wherein saidangled-dual-axis focusing means and said first ends of said first andsecond optical fibers are mechanically coupled to a substrate.
 36. Theapparatus of claim 35 wherein said substrate comprises a siliconsubstrate etched with V-grooves.
 37. The apparatus of claim 1 whereinsaid first and second optical fibers comprises one or more elementsselected from the group consisting of single-mode fibers, polarizationmaintaining fibers, multi-mode fibers, and birefrigent fibers.
 38. Theapparatus of claim 37 wherein each of said first and second opticalfibers comprises a single-mode fiber.
 39. The apparatus of claim 1wherein said observation beam comprises reflected light emanated fromsaid confocal overlapping volume within said sample.
 40. The apparatusof claim 1 wherein said illumination focal volume and said observationfocal volume are diffraction-limited, determined by main lobes of saidillumination beam's point-spread function and said observation beam'spoint-spread function.
 41. The apparatus of claim 40 wherein saidconfocal overlapping volume is diffraction-limited.
 42. The apparatus ofclaim 1 further comprising a frequency shifting means for shifting thefrequency of said observation beam.
 43. The apparatus of claim 42wherein said frequency shifting means is optically coupled to said firstoptical fiber.
 44. The apparatus of claim 42 wherein said frequencyshifting means is optically coupled to said second optical fiber. 45.The apparatus of claim 42 wherein said frequency shifting meanscomprises an element selected from the group consisting of piezoelectricfiber stretchers, electro-optic phase modulators, and acousto-opticfrequency shifters.
 46. The apparatus of claim 1 further comprising afrequency shifting means optically coupled to said reference beam, forshifting the frequency of said reference beam.
 47. The apparatus ofclaim 46 wherein said frequency shifting means comprises an elementselected from the group consisting of piezoelectric fiber stretchers,electro-optic phase modulators, and acousto-optic frequency shifters.48. The apparatus of claim 1 further comprising an optical amplifieroptically coupled to said second optical fiber for amplifying saidobservation beam.
 49. The apparatus of claim 1 further comprising anadjustable optical delay device optically coupled to said first opticalfiber.
 50. The apparatus of claim 1 further comprising an adjustableoptical delay device optically coupled to said second optical fiber. 51.The apparatus of claim 1 further comprising an adjustable optical delaydevice for adjusting an optical path length of said reference beam. 52.The apparatus of claim 1 wherein said illumination beam is a polarizedbeam, and wherein said first and second optical fibers are polarizationmaintaining (PM) fibers capable of supporting two orthogonalpolarizations.
 53. The apparatus of claim 52 further comprising apolarization rotation means optically coupled to said reference beam,for rotating the polarization of said reference beam.
 54. The apparatusof claim 53 wherein said polarization rotation means comprises anelement selected from the group consisting of Faraday rotators,rotatable fiber-optic connectors, and half-wave birefrigent plates. 55.The apparatus of claim 52 further comprising a polarization rotationmeans optically coupled to said second optical fiber, for rotating thepolarization of said observation beam.
 56. The apparatus of claim 55wherein said polarization rotation means comprises an element selectedfrom the group consisting of Faraday rotators, rotatable fiber-opticconnectors, and half-wave birefrigent plates.
 57. The apparatus of claim52 further comprising an auxiliary polarizing beamsplitter opticallycoupled to said second end of said second optical fiber.
 58. Theapparatus of claim 57 further comprising third and fourth PM fibersoptically coupled to said auxiliary polarizing beamsplitter.
 59. Theapparatus of claim 58 further comprising an adjustable optical delaydevice optically coupled to said third PM fiber.
 60. The apparatus ofclaim 58 further comprising an adjustable optical delay device opticallycoupled to said fourth PM fiber.
 61. The apparatus of claim 58 whereinsaid third and fourth PM fibers are joined by a polarization maintainingfiber-optic coupler.
 62. The apparatus of claim 52 further comprising anauxiliary polarizing beamsplitter optically coupled to said referencebeam.
 63. The apparatus of claim 62 further comprising fifth and sixthPM fibers optically coupled to said auxiliary polarizing beamsplitter.64. The apparatus of claim 63 further comprising an adjustable opticaldelay device optically coupled to said fifth PM fiber.
 65. The apparatusof claim 63 further comprising an adjustable optical delay deviceoptically coupled to said sixth PM fiber.
 66. The apparatus of claim 63wherein said fifth and sixth PM fibers are joined by a polarizationmaintaining fiber-optic coupler.
 67. The apparatus of claim 62 furthercomprising a reference PM fiber, wherein a first end of said referencePM fiber is optically coupled to said light-generating means forreceiving said reference beam, and wherein a second end of saidreference PM fiber is optically coupled to said auxiliary polarizingbeamsplitter.
 68. The apparatus of claim 2 wherein said light sourcecomprises an unpolarized light source, wherein said beam-splitting meanscomprises a polarizing beamsplitter, and wherein said first, second andreference optical fibers comprise polarization maintaining fiberscapable of supporting two orthogonal polarizations.
 69. The apparatus ofclaim 2 wherein said light source comprises a polarized light source,wherein said beam-splitting means comprises a polarizing beamsplitter,and wherein said first, second and reference optical fibers comprisepolarization maintaining fibers capable of supporting two orthogonalpolarizations.
 70. The apparatus of claim 2 wherein said light sourcecomprises a polarized light source, wherein said beam-splitting meanscomprises a polarization maintaining fiber-optic coupler, and whereinsaid first, second and reference optical fibers comprise polarizationmaintaining fibers capable of supporting two orthogonal polarizations.71. The apparatus of claim 2 wherein said light source has a coherencelength less than 3000 microns.
 72. The apparatus of claim 2 wherein saidlight source is capable of producing light in the wavelength range of0.8 to 1.6 microns.
 73. The apparatus of claim 7 wherein said lightsource comprises a polarized light source, and wherein said first,second and reference optical fibers comprise polarization maintainingfibers capable of supporting two orthogonal polarizations.
 74. Theapparatus of claim 7 wherein said light source has a coherence lengthless than 3000 microns.
 75. The apparatus of claim 7 wherein said lightsource is capable of producing light in the wavelength range of 0.8 to1.6 microns.
 76. A method for performing angled-dual-axis opticalcoherence scanning microscopy within a sample, comprising: a) producingan illumination beam and a reference beam from a light-generating means,wherein said light-generating means has a predetermined coherencelength; b) routing said reference beam along a reference path; c)transmitting said illumination beam along an illumination path includinga first optical fiber; d) focusing said illumination beam emerging fromsaid first optical fiber to an illumination focal volume along anillumination axis within said sample; e) receiving an observation beamemanated from an observation focal volume along an observation axiswithin said sample, wherein said illumination axis and said observationaxis intersect at an angle within said sample, such that saidillumination focal volume and said observation focal volume intersect ata confocal overlapping volume; f) focusing said observation beamsubstantially into a second optical fiber; g) combining said referencebeam and said observation beam such that coherent interference isproduced; and h) directing said illumination beam and said observationbeam in such a way that said illumination axis and said observation axisremain intersecting at said angle and said confocal overlapping volumescans within said sample, while repeating said step g).
 77. The methodof claim 76 further comprising the step of compensating for the changesin optical path lengths of said illumination and observation beams inthe course of scanning, so as to maintain said coherent interference.78. The method of claim 76 further comprising the step of modulating thephase of said observation beam, such that said coherent interferencebetween said reference beam and said observation beam is modulated at abeat frequency.
 79. The method of claim 76 further comprising the stepof modulating the phase of said reference beam, such that said coherentinterference between said reference beam and said observation beam ismodulated at a beat frequency.
 80. The method of claim 76 furthercomprising the step of shifting the frequency of said observation beam,such that said coherent interference between said reference beam andsaid observation beam is modulated at a beat frequency.
 81. The methodof claim 76 further comprising the step of shifting the frequency ofsaid reference beam, such that said coherent interference between saidreference beam and said observation beam is modulated at a beatfrequency.
 82. The method of claim 76 further comprising the step ofadjusting an optical path length traversed by said observation beam, soas to maintain said coherent interference between said reference beamand said observation beam when combined.
 83. The method of claim 76further comprising the step of adjusting an optical path lengthtraversed by said reference beam, so as to maintain said coherentinterference between said reference beam and said observation beam whencombined.
 84. The method of claim 76 further comprising the step ofoptically amplifying said observation beam.
 85. The method of claim 76further comprising: a) polarizing said reference beam and saidillumination beam such that said reference beam and said illuminationbeam are polarized beams; b) rotating a polarization of said referencebeam relative to said observation beam such that one polarization modeof said observation beam and said reference beam have substantially thesame polarization; and c) combining said reference beam and said onepolarization mode of said observation beam such that coherenceinterference is produced.